Pneumococcal dosing regimen

ABSTRACT

Methods of immunizing older adult subjects against  Streptococcus pneumoniae  infection are provided. Provided methods comprise immunization of naïve adult subjects with a conjugated pneumococcal polysaccharide vaccine. Optionally, initial immunization may be followed by additional immunization doses comprising conjugated pneumococcal polysaccharide vaccine or unconjugated pneumococcal polysaccharide vaccine composition.

PRIORITY INFORMATION

This is a divisional of U.S. application Ser. No. 11/740,580, filed Apr.26, 2007, which claims priority to U.S. Provisional Patent ApplicationSer. No. 60/799,053, filed May 8, 2006, the contents of which are hereinincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is a bacterial pathogen implicated in manyrespiratory and systemic infections, including pneumonia, meningitis,otitis media, bacteremia, sinusitis, peritonitis, and arthritis.Infection with S. pneumoniae can be fatal; other serious results ofinfection can include neurological effects (e.g., learning disabilities,seen in meningitis patients) or hearing loss (in recurrent otitis mediainfections). Young children and the elderly are at a particularly highrisk for infection with S. pneumoniae, as are immuno-compromisedpopulations and members of certain ethnic groups.

Historically, antimicrobial therapies have been useful in reducingmorbidity and mortality rates associated with invasive pneumococcaldisease; however, the increasing prevalence of multi-drug resistantstrains of S. pneumoniae poses troubling difficulties for attempts tocontrol infections through drug therapy alone.

Vaccination efforts have recently proven effective in reduction ofincidence of S. pneumoniae infection rates. Current vaccines containpolysaccharide molecules from the capsule of different serotypes of S.pneumoniae; more than 90 different serotypes have been identified, withdifferent incidences worldwide. Some vaccines, known as “unconjugated”vaccines, contain free polysaccharides. More recently, “conjugated”vaccines, in which saccharide molecules are linked to a peptide carrier,have been developed. An unconjugated vaccine containing polysaccharidesfrom 23 different pneumococcal serotypes has been licensed for use inchildren over 2 years of age; a conjugate vaccine containing 7 differentpolysaccharide valencies has been licensed for pediatric use.Introduction and use of this 7-valent conjugate vaccine, PREVNAR®(produced by Wyeth Pharmaceuticals, Inc. of Philadelphia, Pa. andcontaining polysaccharides from serotypes 4, 6B, 9V, 14, 18C, 19F, and23F conjugated to the Diphtheria CRM₁₉₇ protein), in the United Stateshas reduced the incidence of invasive pneumococcal disease (IPD) inchildren nearly 94% (from 80 per 100,000 in 1998-1999 to 4.6 per 100,000in 2003).

While young children are among those most at risk for S. pneumoniaeinfections, disproportionate morbidity and mortality result frominvasive pneumococcal disease occurring in older adults. The incidenceof invasive pneumococcal infection begins to increase at about age 50years and increases sharply at age 65 years. S. pneumoniae is the mostcommon cause of community-acquired pneumonia in the United States,resulting in hospitalizations and deaths in persons over 65 years(Robinson, K. A. et al., JAMA 285: 1729-35, 2001). Deaths occur inapproximately 14% of patients hospitalized with invasive disease.Persons living in long term care facilities remain a particularlysusceptible population for invasive pneumococcal disease as compared tosimilar older persons living in community settings (Kupronia, B. A., etal., JAGS 51: 1520-1525, 2003).

The only currently licensed pneumococcal vaccine for use in adultpopulations is the 23 valent unconjugated polysaccharide vaccine.Although this vaccine has been recommended by the CDC Advisory Committeeon Immunization Practices (ACIP) Guidelines for prevention ofpneumococcal disease in persons 65 years of age or older, significantcontroversy exists relating to its efficacy. Specifically, widevariation in clinical efficacy has been observed in with this vaccine,which may be related, for example, to age, or disease manifestation(see, e.g., Fedson et al., Arch. Intern. Med., 154: 2531-2535, 1994; andFrench J. Infect., 46: 78-86, 2003). The vaccine appears to beparticularly ineffective in protecting the elderly against pneumococcalpneumonia, and also in protecting any population against otitis mediainfections. Additionally, meta-analyses of randomized controlled studieshave not found a clear protective effect for pneumococcal pneumonia inhigh-risk groups such as elderly populations (See, e.g., Fine, M. J., etal., Arch. Int. Med. 154: 2666-2677, 1994; Moore, R. A., BMC Fam. Prac.1: 1-10, 2000; Conaty, S., et al., Vaccine 22: 3214-3224, 2004; andJackson, L. A., et al., N. Engl. J. Med. 348: 1747-1755, 2003.Furthermore, analyses of immuno-protective effects and duration ofresponse have suggested populations such as the older populations andimmuno-compromised patients may generate only limited immune responsefollowing polysaccharide vaccination, and resulting antibodyconcentrations may decrease rapidly subsequent to vaccination (See,e.g., Davidson, M., et al., Arch. Intern Med. 154: 2209-2214, 1994;Rodriguez-Barradas M. C., J. Infect. Dis. 173: 1347-1353, 1996; Rubins,J. B., J. Infect. Dis. 178: 431-440, 1998; and Sankilampi, U., J.Infect. Dis. 176: 1100-1104, 1997. Moreover, once a subject has beeninitially vaccinated with the unconjugated polysaccharide vaccine,revaccination has been found to result in subsequent hyporesponsiveness.(See, e.g., Artz et al., Clin. Microbiol. Rev. 16: 308-318, 2003;Mufson, M. A., et al., Vaccine 9: 403-407, 1991; Borgondo, J. M., etal., Proc. Soc. Exp. Biol. Med. 157: 148-154, 1978; and Linnemann, G.C., et al., Arch Intern. Med. 146: 1554-1556, 1996.

There remains a need for improved strategies for protecting high riskpopulations, and particularly older and elderly populations, frominfections with S. pneumoniae.

SUMMARY OF THE INVENTION

The present invention encompasses the finding that an initialimmunization of naïve (previously un-immunized) older subjects withpneumococcal conjugate vaccine results in generation of an improvedimmunoprotective response over presently available vaccination. Stillfurther, the invention encompasses the finding that an initialimmunization dose with conjugate vaccine followed by at least oneadditional immunization dose with either conjugate or unconjugatedpolysaccharide vaccine gives a beneficial immunoprotective effect. Thus,provided are methods of immunizing (and re-immunizing) older adultsubjects against pneumococcal infection. Provided methods includemethods for production of antibody titer in naïve adults by immunizingadults with a conjugated pneumococcal polysaccharide vaccine.Alternatively or additionally, provided methods further comprise repeatdosing immunization schedules wherein antibody titers are produced thatexceed levels and functional activity of those observed in prior methodsand studies utilizing pneumococcal polysaccharide vaccines.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

The invention relates to the ability of pneumococcal conjugate vaccinesto increase immunoprotective response generated in older vaccinationsubjects, as compared to currently available vaccination approaches. Forthe purposes of this invention, “pneumococcus” or “pneumococcal” refersto Streptococcus pneumoniae. “Pneumococcal disease” refers to diseasecaused by Streptococcus pneumoniae infection. As described herein,pneumococcal conjugate vaccines are useful in methods of vaccination ofolder subjects against Streptococcus pneumoniae infection and/ordisease. The invention relates to methods of eliciting immunoprotectiveresponse against S. pneumoniae infection in older subjects, includingelderly subjects. Further, the invention relates to methods ofadministering additional dose(s) of pneumococcal vaccine to an oldersubject in order to extend immunoprotection against S. pneumoniaeinfection.

The present invention provides improved methods of vaccination forprevention or amelioration of pneumococcal infection of olderpopulations, including elderly persons. In the context of the inventiona human subject is considered an older subject if s/he is 50 years orover in age. A subject is considered elderly if s/he is 60 years or overin age, typically over 65.

Pneumococcal Vaccines

S. pneumoniae Polysaccharide Antigens

There are at least 90 sub-types of the gram positive S. pneumoniaeorganism, each having a different chemical structure of capsularpolysaccharide. The capsular polysaccharide is the principal virulencefactor of the pneumococcus. It is these capsular polysaccharides which,when isolated, are useful for human induction of antibody response andeffective vaccine generation. Each of the serotype designation as usedherein are designated according to the Danish nomenclature

Typically the Streptococcus pneumoniae vaccine used in the methods ofthe present invention comprises polysaccharide antigens (unconjugated orconjugated to a polypeptide carrier) derived from at least fourpneumococcus serotypes. For example, a vaccine useful in the providedmethods may include at least one polysaccharide selected from serotypes6B, 14, 19F and 23F. In some embodiments, a vaccine comprises at leasttwo or three polysaccharide selected from serotypes 6B, 14, 19F and 23F.In still other embodiments, all four polysaccharide selected ofserotypes 6B, 14, 19F and 23F are included in a vaccine of use in thepresent invention.

In some embodiments, a vaccine useful in the provided methods mayinclude at least one polysaccharide selected from serotypes 4, 6B, 9V,14, 18C, 19F, and 23F. In some embodiments, a vaccine comprises at leasttwo, at least three or at least four polysaccharides selected fromserotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. In still other embodiments,a vaccine comprises at least five, or at least six polysaccharidesselected from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. In certainembodiments, all seven polysaccharides of serotypes 4, 6B, 9V, 14, 18C,19F, and 23F are included in a vaccine used in the provided methods.

In some embodiments, a vaccine useful in the provided methods mayinclude at least one polysaccharide selected from serotypes 1, 4, 5, 6B,9V, 14, 18C, 19F, and 23F. In some embodiments, a vaccine comprises atleast two, at least three, at least four, or at least fivepolysaccharides selected from serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F,and 23F. In still other embodiments, a vaccine comprises at least six,at least seven, or at least eight polysaccharides selected fromserotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F are included in avaccine used in the provided methods. In certain embodiments, all ninepolysaccharides of serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F areincluded in a vaccine used in the provided methods.

In still additional embodiments, a vaccine useful in the methods of theinvention comprises at least one, polysaccharide selected from serotypes1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F. In some embodiments, avaccine comprises at least two or at least three polysaccharidesselected from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. Inother embodiments, a vaccine comprise at least four, at least five, orat least six polysaccharides selected from serotypes 1, 4, 5, 6B, 7F,9V, 14, 18C, 19F and 23F. In still other embodiments, at least seven, atleast eight, or at least nine, polysaccharides are included in a vaccinewhich are selected from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and23F. In certain embodiments, all ten polysaccharides of serotypes 1, 4,5, 6B, 7F, 9V, 14, 18C, 19F and 23F are included in a vaccine used inthe provided methods.

In still additional embodiments, a vaccine useful in the methods of theinvention comprises at least one, polysaccharide selected from serotypes1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F. In some embodiments, avaccine comprises at least two or at least three polysaccharidesselected from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. Inother embodiments, a vaccine comprise at least four, at least five, orat least six polysaccharides selected from serotypes 1, 3, 4, 5, 6B, 7F,9V, 14, 18C, 19F and 23F. In still other embodiments, at least seven, atleast eight, at least nine, or at least ten, polysaccharides areincluded in a vaccine which are selected from serotypes 1, 3, 4, 5, 6B,7F, 9V, 14, 18C, 19F and 23F. In certain embodiments, all elevenpolysaccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C,19F and 23F are included in a vaccine used in the provided methods.

In still additional embodiments, a vaccine useful in the methods of theinvention comprises at least one polysaccharide selected from serotypes1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In someembodiments, a vaccine comprises at least two, at least three or atleast four polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F and 23F. In other embodiments, a vaccinecomprise at least five, at least six, at least seven, or at least eightpolysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F. In still other embodiments, at least nine, atleast eight, at least ten, at least eleven, or at least twelvepolysaccharides are included in a vaccine which are selected fromserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Incertain embodiments, all thirteen polysaccharides derived from serotypes1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are included in avaccine used in the provided methods.

In still further embodiments, a vaccine useful in the methods of theinvention comprises at least one, at least two, at least three, at leastfour, or at least five polysaccharides selected from serotypes 1, 2, 3,4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F,20, 22F, 23F and 33F. In other embodiments, a vaccine comprise at leastsix, at least seven, at least eight, at least nine, or at least tenpolysaccharides selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and33F. In still other embodiments, at least seven, at least eleven, atleast twelve, at least thirteen, at least fourteen, or at least fifteen,polysaccharides are included in a vaccine which are selected fromserotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B,17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In still other embodiments, atleast sixteen, at least seventeen, at least eighteen, at least nineteen,or at least twenty, polysaccharides are included in a vaccine which areselected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In certainembodiments, a vaccine comprises at least twenty one, at least twentytwo, or at least twenty three polysaccharides derived from serotypes 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,19A, 19F, 20, 22F, 23F and 33F. In yet another embodiment, the inventionmethod contemplates use of vaccine comprising at least 23 polysaccharideantigens derived of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A,11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In certainembodiments, the invention method contemplates use of vaccine comprisingall 23 polysaccharide antigens derived of serotypes 1, 2, 3, 4, 5, 6B,7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23Fand 33F.

Although the above polysaccharides may be used in their full-length,native form, it should be understood that size-reduced polysaccharidesmay also be used which are still immunogenic (see for example EP 497524and 497525).

Polysaccharide Vaccines

As discussed above, isolated capsular polysaccharides are useful vaccineantigens. Multiple type specific capsular polysaccharides have beenprepared individually, and combined for generation of a vaccine which iscapable of eliciting an immune response against multiple pneumococcalstrains. Such vaccine generations are well known as unconjugatedpneumococcal polysaccharide vaccine preparations. Two differentunconjugated pneumococcal vaccines have been reported and approved foradministration to humans. The first unconjugated polysaccharidepneumococcal vaccine comprised a 14-valent unconjugated pneumococcalpolysaccharide vaccine; the second, currently in use, was a 23-valentunconjugated pneumococcal polysaccharide vaccine.

The currently available 23 valent polysaccharide vaccine includespolysaccharide from the following serotypes: 1, 2, 3, 4, 5, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and33F, accounting for about 90% of pneumococcal blood isolates presentlyidentified as relevant for infection. In principle, a polysaccharidevaccine could be modified so as to include additional and/or differentcombinations of serotype specific polysaccharides. Modification and/oraddition of polysaccharides may be desirable for example, as infectiousserotypes may shift over time, and/or various serotypes may beparticularly relevant for specific populations.

Currently, the 23-polyvalent polysaccharide vaccine (such asPNEUMOVAX®23, Merck, Whitehouse Station, N.J., also PNEUMOVAX®II,Aventis Pasteur MSD, Berkshire, UK) is available for adults and childrenover two years of age. Each recommended dose of vaccine (0.5 ml)contains 25 mg of each of 23 type-specific polysaccharides dissolved inisotonic saline. The vaccine is often generally referred to as 23vPSvaccine or 23vPS to reflect 23 valence and free polysaccharide (PS).PNEUMOVAX®23 is described at pages 1768-1770 of the 1997 edition of thePhysician's Desk Reference (Medical Economics, Montvale, N.J.).PNEUMOVAX®23 is prepared by a process that involves purifying the abovedescribed 23 polysaccharides from S. pneumoniae, followed by dissolvingthe purified polysaccharides in isotonic saline containing 0.25% phenolas a preservative. The 23-valent polysaccharide vaccine, and productionof the saccharides is described in U.S. Pat. No. 4,686,102.

In general, a variety of methods for preparing unconjugated pneumococcalpolysaccharide vaccines suitable for use in the present invention areknown in the art. In many cases, polysaccharides are purifiedindividually from S. pneumoniae serotypes. In general, a starting pointin the entire process of making the vaccine is the purification ofbacterial capsular polysaccharides from a fermentation broth of aparticular serotype of S. pneumoniae. Alternatively or additionally,polysaccharides may be isolated simultaneously from more than one S.pneumoniae serotype, so that a mixture of serotype polysaccharides isobtained and the relevant serotype polysaccharides are already combined.In some instances, different polysaccharides may require sufficientlydifferent isolation procedures that such simultaneous isolation is notdesirable or is not feasible. Methods of purifying polysaccharides fromS. pneumoniae are well known. For example, a purification process forthe polysaccharides is described in U.S. Pat. Nos. 4,686,102; 4,242,501;4,221,906; 5,623,057; and 5,847,112. Preparation of the 23-valentpolysaccharides has been described in U.S. Pat. No. 4,686,102.

Alternatively, pneumococcal polysaccharides are also commerciallyavailable (e.g., available from ATCC), and may be used as startingmaterial for vaccine preparation. Individual native polysaccharides (ina powder form) are dissolved in water, and incubated with a salt (e.g.,sodium chloride) to dissociate residual impurities which are thenremoved by filtration. Purified polysaccharides can then be dissolved inan appropriate solvent or buffer (e.g., isotonic saline), optionallycontaining a preservative (e.g., 0.25% phenol).

Resulting purified polysaccharides can then be used, eitherindividually, or in combination for production of a vaccine. Methods ofpreparation of unconjugated vaccines that are suitable for use in thepresent invention are described in, e.g., U.S. Pat. Nos. 4,242,501,4,221,906 and 4,686,102, and European Patent Application EP 0 002 404.Preparation of the 23-valent polysaccharides has been described in U.S.Pat. No. 4,686,102.

Conjugate Vaccines

Conjugate vaccines are prepared by linking isolated or purifiedpolysaccharides with a polypeptide carrier. It is generally believedthat conjugate vaccines promote stronger T-cell-dependent immuneresponses than do unconjugated polysaccharides. In particular, conjugatevaccines have proven to be more strongly immunogenic in children undertwo years of age; such young children do not mount a strong response tomost unconjugated polysaccharides.

In general, any polypeptide carrier can be used that allows coupling ofpolysaccharide antigens so that they are displayed in a way that inducesan immunoprotective immune response. More than one polysaccharideantigen may be coupled to the same polypeptide carrier molecule orentity. For instance, two or more different polysaccharide antigens(whether first isolated separately and then combined or isolatedtogether) may be simultaneously coupled to polypeptide carrier in thesame reaction. Or, a polypeptide carrier that has already been coupledwith one polysaccharide antigen can subsequently be coupled withanother. In other embodiments, individual polypeptide carrier moleculesor entities are each coupled to only one polysaccharide antigen. Forexample, individual isolated antigens may be separately coupled topolypeptide carriers and then combined with one another after coupling.All polysaccharide antigens in a particular conjugate vaccine may becoupled to the same polypeptide carrier, or alternatively differentpolypeptide carriers may be employed.

Polypeptide Carriers

In many embodiments of the invention, conjugate vaccines will comprisepolysaccharide antigens coupled to one or more polypeptide carriers. Inprinciple, any polypeptide suitable for presenting pneumococcalpolysaccharide antigens such that an immunoprotective immune response tothe polysaccharide antigen is induced may be utilized in accordance withthe present invention.

A polypeptide carrier can be used, In general a polypeptide carrier mayhave at least about 50 amino acid residues in the chain, preferably100-1000 amino acid residues. In certain instances, it may be desirablethat the polypeptide carrier have at least some lysine residues orglutamate or aspartate residues of the amino acids sequence. The pendantamines of lysine residues and pendant carboxylates of glutamine andaspartate are convenient for attaching an agent (e.g., polysaccharide).

Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate. Additionally suitable polypeptide carriers include bacterialtoxins, toxoids, or inactivated toxin. A toxoid is a bacterial toxinwhose toxicity has been weakened or suppressed while other properties,typically immunogenicity, are maintained. As a class, bacterial toxinsand derivatives thereof tend to be highly immunogenic. Polypeptidecarriers derived from bacterial toxins have proven to be effectivepolypeptide carriers in vaccines. Generally, steps are taken (e.g., bychemical and/or genetic means) to render the toxins non-toxic and safefor administration to mammals. Examples of such bacterial toxin-derivedpolypeptide carriers which are currently commonly used in vaccinecompositions, and may be used in conjugate antigens of vaccines usefulin the provided methods, include the diphtheria and tetanus toxoids, andvariants thereof (e.g., DT, DT CRM₁₉₇, TT), cholera toxoid, pertussistoxoid, inactivated or mutant pneumococcal pneumolysin, pneumococcalsurface protein A or a derivative thereof, pneumococcal adhesion proteinA or a derivative thereof, C5a peptidase group a or group bstreptococcus or a derivative thereof, non-typable H. influenzae P4protein or a derivative thereof, non-typable H. influenzae P6 protein ora derivative thereof, M. catarrhalis uspA or a derivative thereof,keyhole limpet haemocyanin (KLH), OMPC from N. meningitidis, thepurified protein derivative of tuberculin (PPD), and protein D fromHaemophilus influenzae (EP 594610-B), or fragments of any of theforegoing. Fragments suitable for use include fragments encompassingT-helper epitopes, which are readily known or identified by one skilledin the art.

CRM₁₉₇ (Wyeth, Sanford, N.C.) is a non-toxic variant (i.e., toxoid) ofdiphtheria toxin isolated from cultures of Corynebacterium diphtheriastrain C7 (β197) grown in casamino acids and yeast extract-based medium.CRM₁₉₇ is purified through ultra-filtration, ammonium sulfateprecipitation, and ion-exchange chromatography. Alternatively, CRM₁₉₇ isprepared recombinantly in accordance with U.S. Pat. No. 5,614,382, whichis hereby incorporated by reference. Other diphtheria toxoids are alsosuitable for use as carrier proteins.

Conjugation Methods

A variety of strategies for coupling polysaccharide antigens topolypeptide carriers are known in the art. For example, each of thefollowing references discusses relevant methods: Dick and Burret,Contrib Microbiol Immunol. 10:48-114 (Cruse J M, Lewis R E Jr, eds;Basel, Krager, 1989); U.S. Pat. No. 4,372,945; U.S. Pat. No. 4,474,757;U.S. Pat. No. 4,673,574; U.S. Pat. No. 4,695,624; U.S. Pat. No.4,882,317; U.S. Pat. No. 5,360,897; U.S. Pat. No. 5,371,197; U.S. Pat.No. 5,623,057; European Patent Application EP 0 497 524; andInternational Patent Publication WO 94/04195.

In some embodiments polysaccharide antigens are coupled to polypeptidecarriers using CDAP (see, for example, WO 95/08348).

Existing Conjugated Vaccines

Pneumococcal conjugate vaccine compositions useful in the practice ofthe present invention have been prepared in the art. To name just a fewexamples:

Two different heptavalent conjugate vaccines have been developed thatcontain capsular polysaccharides from pneumococcal serotypes 4, 6B, 9V,14, 18C, 19F, and 23F. Such vaccines are known as “7vPnC” vaccinesbecause they contain 7 valencies (7v) of the pneumococcal polysaccharide(Pn) as a conjugate (C). The first of these 7vPnC vaccines was PREVNAR®,developed by Wyeth Pharmaceuticals, Inc. (Philadelphia, Pa.). InPREVNAR®, the polysaccharides are each conjugated to the non-toxicCRM₁₉₇ variant of diphtheria toxin. PREVNAR® is the first pneumococcalconjugate vaccine approved for use in humans in the United States, andis currently licensed for pediatric use. Description of the heptavalentconjugate vaccine, and production of the conjugates is described in U.S.Pat. Nos. 4,673,574, 4,902,506, and 5,360,897.

The other 7vPnC vaccine was generated and reported by Merck & Co., WestPoint, Pa. In this vaccine, the 7 capsular polysaccharides areconjugated to the outer membrane protein complex (OMPC) of Neisseriameningitidis (Merck 7vPnC-OMPC).

Wyeth reported a nonavalent conjugated pneumococcal vaccine, containingcapsular polysaccharides from pneumoccal serotypes 1, 4, 5, 6B, 9V, 14,18C, 19F and 23F, each conjugated to the non-toxic CRM₁₉₇ variant ofdiphtheria toxin (Huebner, R., et al., Ped. Inf. Dis. J., 21:1004-1007,2002). (Wyeth, 9vPnC-MnCC).

Furthermore, a 13-valent pneumococcal vaccine has been generated byWyeth, which comprises the capsular polysaccharides 4, 6B, 9V, 14, 18C,19F, 23F, 1, 3, 5, 7F, 6A, and 19A conjugated to CRM₁₉₇ variant ofdiphtheria toxin (U.S. Ser. No. 11/395,593, filed Mar. 31, 2006).

Two tetravalent conjugated pneumococcal vaccines have also beengenerated and reported by Sanofi-Aventis, Swiftwater, Pa. The firstcontains capsular polysaccharides of serotypes 6B, 14, 19F, and 23Findividually conjugated to diphtheria toxoid (Aventis 4vPnD); and thesecond is the same polysaccharides conjugated to tetanus toxoid (Aventis4vPnT).

GlaxoSmithKline has reported an eleven-valent conjugated pneumococcalvaccine, containing capsular polysaccharides from a different set ofserotypes, namely 6B, 14, 19F, 23F, 1, 3, 4, 5, 7F, 9V, and 18C. Thesepolysaccharide antigens are individually conjugated to protein D of H.influenzae (GlaxoSmithKline, 11vPnC D).

GlaxoSmithKline has also reported a ten-valent conjugated pneumococcalvaccine, containing capsular polysaccharides from serotypes 1, 4, 5, 6B,7F, 9V, 14, 18C, 19F, and 23F. These polysaccharide antigens areindividually conjugated to protein D of H. influenzae (GlaxoSmithKline,10vPnC D).

Sanofi-Aventis has reported generation of yet additional eleven-valentconjugated pneumococcal vaccines, containing polysaccharide antigensfrom the same serotype set found in the Merck & Co., product (Merck11vPnC OMPC). A first has been conjugated to tetanus toxoid (Aventis11vPnC T, and another contains polysaccharide conjugated to twodifferent polypeptide carriers. Specifically, capsular polysaccharidesfrom serotypes 3, 6B, 14, and 18C are conjugated to diphtheria toxoid,whereas capsular polysaccharides from serotypes 1, 4, 5, 7F, 9V, 19F,and 23F are conjugated to tetanus toxoid (Aventis 11vPnCTD).

Sanofi-Aventis also has also reported generation of two differentversions of an eight-valent conjugate pneumococcal vaccine, containingcapsular polysaccharides of serotypes 3, 6B, 14, 19F, 4, 23F, 9V, and18C. In one, the polysaccharide antigens are conjugated to diphtheriatoxoid; in the other they are conjugated to tetanus toxoid (Aventis8vPnC D or 8vPnCT.).

Immunization Schedule

According to the present invention, vaccine administration may involvedelivery of only a single dose, or alternatively may involve an initialdose followed by one or several additional immunization doses,adequately spaced. An immunization schedule is a program for theadministration of one or more specified doses of one or more specifiedpneumococcal vaccines, by one or more specified routes ofadministration, at one or more specified ages of a subject.

The present invention provides immunization methods that involveadministering at least one dose of a pneumococcal conjugate vaccine toan adult subject. In some embodiments, the adult subject is older thanabout 50 years of age. In some embodiments the adult subject is olderthan about 65 years of age. In some embodiments, the adult subject haspreviously received one or more doses of an unconjugated pneumococcalpolysaccharide vaccine; in other embodiments, the adult subject is naïveto pneumococcal vaccines. In some embodiments, the adult subject haspreviously been infected with, or exposed to infection by S. pneumoniae.In some embodiments of the present invention, the subject will receiveone or more additional doses of a pneumococcal vaccine, which may be aconjugate vaccine or an unconjugated vaccine; in other embodiments, thesubject will not receive further pneumococcal vaccinations.

As illustrated in the Exemplification, the present inventiondemonstrates that pneumococcal conjugate vaccines can induce a superiorincrease in antibody generation, and a superior functional antibodyresponse in older subjects when compared with an unconjugatedpolysaccharide vaccine. In particular, when the 7vPnC conjugate vaccinewas administered to vaccine-naïve individuals over 70 years of age, bothantibody titers (measured by ELISA) and functional antibody levels(measured by OPA) were higher for 6 of 7 common serotypes than wereobserved with the 23vPS unconjugated polysaccharide vaccine.Furthermore, studies presented in the Exemplification confirmed resultsseen in other studies where an initial administration of polysaccharidevaccine (e.g., 23vPS) appears to induce hyporesponsiveness to subsequentvaccine administration, as measured by decreased antibody level orfunctional antibody responses to subsequent polysaccharide (e.g., 23vPS)or conjugate vaccine (e.g., 7vPnC) administration, while this is notseen after PnC vaccine.

Immunization schedules of the present invention are provided to elicitor induce an immune response (e.g., an immuno-protective response) in anolder subject sufficient to reduce at least one measure selected fromthe group consisting of incidence, prevalence, frequency and severity ofat least one pneumococcal disease or disorder, and/or at least onesurrogate marker of the disorder, in a population and/or subpopulationof the subject(s). A supplemental immunization schedule is one which hasthis effect relative to the standard schedule which it supplements. Asupplemental schedule may call for additional administrations and/orsupraimmunogenic doses of polysaccharide(s) and/or conjugate(s) found inthe standard schedule, or for the administration of vaccines not part ofthe standard schedule. A full immunization schedule of the presentinvention may comprise both a standard schedule and a supplementalschedule. Exemplary sample vaccination schedules are provided forillustrative purposes. Detailed descriptions of methods to assessimmunogenic response discussed herein allow one to develop alterationsto the sample immunization schedules without undue experimentation.

In one embodiment of the present invention, a first administration of apneumococcal vaccine usually occurs when a subject is more than about 50years old, more than about 55 years old, more than about 60 years old,more than about 65 years old, or more than about 70 years old.

In some embodiments of the invention, a single administration ofconjugate vaccine is employed. It is possible that the purposes of thepresent invention can be served with a single administration, especiallywhen one or more utilized vaccine polysaccharide(s) and/or conjugate(s)or combinations thereof is/are strong, and in such a situation a singledose schedule is sufficient to induce a lasting immunoprotectiveresponse.

In certain embodiments, it is desirable to administer two or more dosesof pneumococcal vaccine, which may include one dose of conjugate and atleast one additional dose of conjugate and/or unconjugatedpolysaccharide vaccine for greater immunoprotective efficacy andcoverage. Thus, in some embodiments, a number of doses is at least two,at least three or more doses. There is no set maximum number of doses,however it is good clinical practice not to immunize more often thannecessary to achieve the desired effect.

Without being bound by theory, a first dose of pneumococcal conjugatevaccine administered according to the invention may be considered a“priming” dose. In certain embodiments, more than one dose is includedin an immunization schedule. In such a scenario, a subsequent dose maybe considered a “boosting” dose.

A priming dose may be administered to a naïve subject (a subject who hasnever previously received a pneumococcal polysaccharide vaccine, or aconjugate vaccine). In some embodiments, a priming dose may beadministered to a subject who has previously received polysaccharidevaccine at least five or more years previous to administration of aninitial conjugate vaccine according to the invention. In otherembodiments, a priming dose may be administered to a subject who haspreviously received conjugate vaccine at least twenty or more yearsprevious to administration of a priming conjugate vaccine according tothe invention.

A boosting dose may be administered to an older subject who haspreviously received a conjugate vaccine. A boosting dose may compriseadministration of a vaccine composition which is identical to thepreviously received priming dose. Alternatively and/or additionally, aboosting dose may comprise administration of a vaccine composition whichis distinct from the previously received priming dose. In someembodiments, a boosting dose comprises at least one of the conjugate(s)of the previously received priming dose, and further comprises one ormore additional conjugate(s) which were not contained in the primingdose. In other embodiments, a boosting dose comprises at least some ofthe conjugate(s) of the previously received priming dose, and furthercomprises one or more additional polysaccharide(s) which were notcontained in the priming dose. In still other embodiments, a boostingdose comprises polysaccharide(s) which were not contained in the primingdose, and the boosting dose does not comprise conjugate(s) which werecontained within the priming dose. Any number of boosting doses may beindicated to maintain an immunoprotective effect against S. pneumoniaeinfection.

When an immunization schedule calls for two or more separate doses, theinterval between doses is considered. The interval between twosuccessive doses may be the same throughout an immunization schedule, orit may change as the subject ages. In immunization schedules of thepresent invention, once a first vaccine dose has been administered,there is a first interval before administration of a subsequent dose. Afirst interval is generally at least about two weeks, one month, sixweeks, two months, three months, six months, nine months, 12 months, orlonger. Where more than one subsequent dose(s) are administered, second(or higher) intervals may be provided between such subsequent doses. Insome embodiments, all intervals between subsequent doses are of the samelength; in other embodiments, second intervals may vary in length. Insome embodiments, the interval between subsequent doses may be at leastabout twelve months, at least about fifteen months, at least abouteighteen months, at least about twenty-one months or at least about twoyears. In certain embodiments, the interval between doses may be up tothree years, up to about four years, or up to about five years or tenyears or more. In certain embodiments, intervals between subsequentdoses may decrease as the subject ages.

The timing of the initial dose and the intervals between successivedoses together determine how many doses occur within a particular periodin a subject's life.

As noted above, exemplary sample schedules are provided herein. SampleSchedule 1 is an intensive immunization schedule where a recipient isimmunized after about age 50 and receives subsequent pneumococcalimmunizations every year. In addition to pneumococcal vaccination, anadditional vaccine may be optionally included for administration (e.g.,influenza vaccination). All vaccines are administered during eachscheduled immunization. Vaccines may be continued for differentdurations of time.

Sample Schedule 2 is another possible immunization schedule Immunizationis initiated after about age 50. As in Schedule 1, vaccination may beoptionally continued or omitted for different durations of time.

Sample Schedule 3 is a third possible immunization schedule. Vaccinationis initiated in a subject after about age 50 and immunization continuesevery five years. After about age 60 subsequent, immunization dosing maybe administered at about every two years. After about age 65, subsequentimmunization may be administered at about every year. Additionalvaccines may be optionally included (e.g., influenza vaccine), andvaccines may be added or omitted or continued for different durations oftime.

Sample Schedule 4 is yet another possible immunization schedule.Vaccination of a subject may have been previously initiated withpolysaccharide (unconjugated) pneumococcal vaccine. In this schedule,vaccination is re-initiated with a conjugate vaccine after about age 60,or after about age 65. An initial dose of conjugate vaccine may beadministered after about five years following an initial dose ofpneumococcal polysaccharide vaccine. Optionally, subsequent vaccinedoses may be included. Subsequent vaccine doses may be administered atabout two years following the initial prime dose, or at about one yearfollowing the initial priming dose, depending on the subject. In someaspects, a subject may continue according to one of Schedules 1-3described above after initial re-priming with conjugate vaccine.

It will be appreciated by those skilled in the art that a variety ofpossible combinations and subcombinations of the various conditions oftiming of the first administration, shortest interval, largest intervaland total number of administrations (in absolute terms, or within astated period) exist, and all of these combinations and subcombinationsshould be considered to be within the inventor's contemplation thoughnot explicitly enumerated here.

Assays for Determination of Immunogenic Response

It may often be desirable to assess the immunological response orresponses achieved in subjects who receive one or more pneumococcalvaccine administrations according to the present invention. Any of avariety of methods may be used in such assessments.

Generally speaking, it may be desirable to assess humoral responses,cellular responses, and/or interactions between the two. Where humoralresponses are being assessed, antibody titers and/or types (e.g., totalIgG, IgG1, IgG2, IgM, IgA, etc.) to specific pneumococcal serotypes maybe determined, for example before and/or after administration of aninitial or a boosting dose of vaccine (and/or as compared with antibodylevels in the absence of antigenic stimulation). Cellular responses maybe assessed by monitoring reactions such as delayed typehypersensitivity responses, etc. to the carrier protein. Precursor andmemory B cell populations may be assessed in ELISpot assays directedagainst specific pneumococcal capsular polysaccharides.

Any of a variety of assays may be employed to detect levels and/oractivity of antibodies in subject sera. Suitable assays include, forexample, ligand binding assays, such as radioimmunoassay (RIAs),enzyme-linked immunosorbent assays (ELISAs), and multiplex assays(Luminex, Bioplex); functional assays, such as opsonophagocytic assays(OPA); and in vivo protection assays (infant rat protection and adultmouse lung colonization and mortality models).

The RIA method detects type specific antibodies through incubation ofsera with radiolabeled type-specific polysaccharides in suspension (see,e.g., Schiffiman et al., J. Immunol Meth. 33:133-144. 1980). Theantigen-antibody complexes are then precipitated with ammonium sulfateand the radiolabeled pellets assayed for counts per minute (cpm).

In the ELISA detection method, serotype-specific antibodies from thesera of vaccinated subjects are quantitated by incubation withserotype-specific polysaccharides which have been adsorbed to a solidsupport (see, e.g., Koskela & Leinonen, J. Clin. Pathol. 34:93-98, 1981;Kojima et al., 1990, Tohoku J. Exp. Med. 161:209-215, 1990; Concepcionand Frasch, 2001. Clin Lab Diagn Immunol 8 266-272). The bound antibodyis detected using enzyme-conjugated secondary detection antibodies. TheELISA also allows isotyping and subclassing of the immune response(i.e., IgM vs. IgG or IgG1 vs. IgG2) by using isotype- orsubclass-specific secondary antibodies and can be adapted to evaluatethe avidity of the serotype-specific antibodies (Anttila, et al, 1998.J. Infect Dis. 177:1614; Romero-Steiner, et al. 2005. Clin Lab DiagnImmunol 12: 1029-1035) Multiplex assays (e.g., Luminex, Bioplex) enablethe simultaneous detection of antibodies to multiple serotypes.Serotype-specific capsular polysaccharides are conjugated to spectrallydistinct microspheres, which are mixed together and incubated withdiluted serum. Bound antibody is detected with aphycoerithrin-conjugated secondary antibody and is quantitated in aspecialized flow cytometer that uses one laser to identify the bead type(serotype) and a second laser to quantitate the bound secondary antibody(Pickering, et al, 2002. Am. J, Clin. Pathol. 117:589-596; Lal, et al.2005. J. Immunol Methods 296: 135-147).

An approach for assessing functional antibody in serum is theopsonophagocytic assay (OPA)) which quantitates only the antibodies thatcan opsonize the bacteria, leading to ingestion and killing of thebacteria. The standard assay utilizes a human phagocytic effector cell,a source of complement, encapsulated pneumococci, and diluted sera. Theassay readout is the serum endpoint titer at which ≧50 of the input CFUs(i.e., bacteria) are killed in the assay (Romero-Steiner, et al, 1997.Clin. Diagn. Lab. Immunol 4. 415-422) This killing OPA can also bemultiplexed by utilizing target strains of pneumococci that carrydifferent antibiotic resistance markers (Kim, et al, 2003. Clin LabDiagn Immunol. 10: 616-621). An endpoint titer of 1:8 or greater isconsidered a positive result in these killing type OPA. Another type ofmultilplex opsonic assay is a nonkilling assay in which the uptake byphagocytic effector cells of fluorescent stained encapsulatedpneumococci or fluorescent microspheres conjugated with pneumocococcalcapsular polysaccharides in the presence of diluted sera plus acomplement source is evaluated by flow cytometry (Martinez, et al, 1999.Clin Lab Diagn Immunol. 6: 581-586). Opsonic activity of serum antibodyplus complement can also be evaluated by measuring the oxidativeresponse of phagocytic human effector cells to ingested pneumococci(Munro, et al. 1985. Clin Exp Immunol 61: 183-188; Ojo-Amaize, et al.1995. Clin Lab Diagn Immunol 2: 286-290).

Certain in vivo model systems can be used to evaluate the protectionafforded by serum antibodies elicited by pneumococcal vaccines. In suchpassive protection systems, mice or infant rats are challenged withencapsulated pneumococci plus diluted human sera, and the endpoint titerof the sera which provides protection against bacteremia, lungcolonization, or mortality is determined (Stack, et al. 1998. J InfectDis. 177: 986-990; Saeland, et al. 2000. Microb Pathog 29: 81-91).

Vaccine Compositions and Administration

According to the present invention, pneumococcal vaccines are used toprotect or treat a subject susceptible to or suffering from S.pneumoniae infection. In certain embodiments, the subject is an oldersubject. In some embodiments, the subject is an elderly subject. In someembodiments, the subject is naïve.

Any individual who suffers from a pneumococcal disease or who is at riskof developing a pneumococcal disease may be treated. It will beappreciated that an individual can be considered at risk for developinga disease without having been diagnosed with any symptoms of thedisease. For example, if the individual is known to have been, or to beintended to be, in situations with relatively high risk of exposure topneumococcal infection, that individual will be considered at risk fordeveloping the disease (e.g., an elderly subject living in long termcare facility). Similarly, if members of an individual's family orfriends, or other close contacts have been diagnosed with pneumococcalinfection, the individual may be considered to be at risk for developingthe disease. Other exposures can include crowding, cigarette smoking,indoor smoke exposure ie wood burning stoves associated with certaingroups, eg American Indians, etc).

Any effective route of administration may be utilized such as, forexample, orally, nasally, enterally, parenterally, intramuscularly orintravenously, subcutaneously, intradermally, rectally, vaginally,topically, ocularly, pulmonarily, or by contact application. In someembodiments, vaccine compositions may be injected (e.g., viaintramuscular, intraperitoneal, intradermal and/or subcutaneous routes);or delivered via the mucosa (e.g., to the oral/alimentary, respiratory,and/or genitourinary tracts). Intranasal administration of vaccines maybe particularly useful in some contexts, for example for treatment ofpneumonia or otitis media (as nasopharyngeal carriage of pneumococci canbe more effectively prevented, thus attenuating infection at itsearliest stage). In some embodiments of the invention, it may bedesirable to administer different doses of a vaccine by differentroutes; in some embodiments, it may be desirable to administer differentcomponents of one dose via different routes.

In some embodiments of the present invention, vaccines are administeredintradermally. Conventional technique of intradermal injection, the“mantoux procedure”, comprises steps of cleaning the skin, and thenstretching with one hand, and with the bevel of a narrow gauge needle(26-31 gauge) facing upwards the needle is inserted at an angle ofbetween 10-15°. Once the bevel of the needle is inserted, the barrel ofthe needle is lowered and further advanced while providing a slightpressure to elevate it under the skin. The liquid is then injected veryslowly thereby forming a bleb or bump on the skin surface, followed byslow withdrawal of the needle.

Devices that are specifically designed to administer liquid agents intoor across the skin have been described, for example the devicesdescribed in WO 99/34850 and EP 1092444, also the jet injection devicesdescribed for example in WO 01/13977; U.S. Pat. No. 5,480,381, U.S. Pat.No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat. No. 5,993,412, U.S.Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S. Pat. No. 5,704,911,U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,893,397, U.S. Pat. No.5,466,220, U.S. Pat. No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat.No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat. No. 5,520,639, U.S.Pat. No. 4,596,556, U.S. Pat. No. 4,790,824, U.S. Pat. No. 4,941,880,U.S. Pat. No. 4,940,460, WO 97/37705 and WO 97/13537. Other methods ofintradermal administration of the vaccine preparations may includeconventional syringes and needles, or devices designed for ballisticdelivery of solid vaccines (WO 99/27961), or transdermal patches (WO97/48440; WO 98/28037); or applied to the surface of the skin(transdermal or transcutaneous delivery WO 98/20734; WO 98/28037).

As described above, vaccines may be administered as a single dose or asmultiple doses. It will be appreciated that an administration is asingle “dose” so long as all relevant components are administered to asubject within a window of time; it is not necessary that everycomponent be present in a single composition. For example,administration of two different antigen (e.g., polysaccharide(s), orconjugate(s), or combination(s) thereof, or of two packets of the sameantigen, within a period of less than 24 hours, is considered a singledose. To give but one example, polysaccharide antigens (or conjugates)from different pneumococcal serotypes may be administered in differentcompositions, but as part of a single dose. As noted above, suchdifferent compositions may be administered via different routes or viathe same route. Alternatively or additionally, in embodiments wherein avaccine comprises combination of polysaccharide antigen(s) (orconjugates) and additional types of active agents, polysaccharide (orconjugate) may be administered via one route, and a second active agentmay be administered by a different route.

Vaccine compositions are administered in such amounts and for such timeas is necessary to achieve a desired result. In certain embodiments ofthe present invention, a vaccine composition comprises an immunogenicamount of at least one pneumococcal polysaccharide or conjugate. As usedherein, an “immunogenic” amount of the vaccine composition is an amountwhich is suitable to elicit an immune response. Thus, the amounteffective to treat, attenuate, or prevent disease, as used herein,refers to a nontoxic but sufficient amount of the vaccine composition totreat, attenuate, or prevent disease in any subject. For example, the“therapeutically effective amount” can be an amount to treat, attenuate,or prevent infection (e.g., bacterial infection, pneumococcalinfection), etc. The exact amount required to achieve a “immunogenicamount” may vary, depending on the particular component (e.g.,polysaccharide, conjugate), and from subject to subject, depending onthe species, age, and general condition of the subject, the stage of thedisease, the particular pharmaceutical mixture, its mode ofadministration, and the like.

The amount of polysaccharide(s) antigen or conjugate(s) in each vaccinedose is selected to allow the vaccine, when administered as describedherein, to induce an appropriate immunoprotective response withoutsignificant, adverse side effects A “immuno-protective” or “protectiveimmune” response as used herein is an immune response sufficient toprotect an immunized subject from productive infection by a particularpathogen or pathogens to which a vaccine is directed (e.g., S.pneumoniae infection). Such amounts may vary depending upon whichspecific polysaccharide(s) or conjugate(s), or combinations ofpolysaccharide(s) and/or conjugate(s) is employed and how it ispresented. Generally, it is expected that each dose will comprise0.1-100 μg of polysaccharide, about 0.1-50 μg, about 0.1-10 μg, or about1 to 5 μg.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. Following an initial vaccination, subjectscan receive one or several booster immunizations adequately spaced intime.

Pneumococcal polysaccharide antigens (or conjugates thereof), and/orpreparations thereof may be formulated in a unit dosage form for ease ofadministration and uniformity of dosage. The expression “unit dosageform,” as used herein, refers to a physically discrete unit of vaccinecomposition appropriate for the patient to be treated. The specifictherapeutically effective dose level for any particular patient ororganism may depend upon a variety of factors including the severity ordegree of risk of infection; the activity of the specific vaccine orvaccine composition employed; other characteristics of the specificvaccine or vaccine composition employed; the age, body weight, generalhealth, sex of the subject, diet of the subject, pharmacokineticcondition of the subject, the time of administration (e.g., with regardto other activities of the subject such as eating, sleeping, receivingother medicines including other vaccine doses, etc.), route ofadministration, rate of excretion of the specific vaccine or vaccinecomposition employed; vaccines used in combination or coincidental withthe vaccine composition employed; and like factors well known in themedical arts.

Pneumococcal vaccines for use in accordance with the present inventionmay be formulated according to known techniques. Vaccine preparation isgenerally described in Vaccine Design (“The subunit and adjuvantapproach” (eds Powell M. F. & Newman M. J., Plenum Press New York,1995). For example, an immunogenic amount of a vaccine product can beformulated together with one or more organic or inorganic, liquid orsolid, pharmaceutically suitable carrier materials. Preparation ofpneumococcal polysaccharide and conjugate vaccines is described, forexample, in U.S. Ser. No. 11/395,593, filed Mar. 31, 2006, the contentsof which are incorporated herein by reference.

In general, pharmaceutically acceptable carrier(s) include solvents,dispersion media, and the like, which are compatible with pharmaceuticaladministration. For example, materials that can serve aspharmaceutically acceptable carriers include, but are not limited tosugars such as lactose, glucose, dextrose, and sucrose; starches such ascorn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; polyolssuch as glycerol, propylene glycol, and liquid polyethylene glycol;esters such as ethyl oleate and ethyl laurate; agar; buffering agentssuch as magnesium hydroxide; alginic acid; pyrogen-free water; isotonicsaline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as preservatives,and antioxidants can also be present in the composition, according tothe judgment of the formulator (see also Remington's PharmaceuticalSciences, Fifteenth Edition, E. W. martin (Mack Publishing Co., EastonPa., 1975).

As is well known, vaccines may be formulated by combining polysaccharideantigens (and/or conjugates) with carriers and/or other optionalcomponents by any available means including, for example, conventionalmixing, granulating, dissolving, lyophilizing, or similar processes.

Vaccine compositions useful in the provided methods may be lyophilizedup until they are about to be used, at which point they areextemporaneously reconstituted with diluent. In some embodiments,vaccine components or compositions are lyophilized in the presence ofone or more other components (e.g., adjuvants), and are extemporaneouslyreconstituted with saline solution. Alternatively, individualcomponents, or sets of components may be separately lyophilized and/orstored (e.g., in a vaccination kit), the components being reconstitutedand either mixed prior to use or administered separately to the subject.

Lyophilization can produce a more stable composition (for instance bypreventing or reducing breakdown of polysaccharide antigens.Lyophilizing of vaccines or vaccine components is well known in the art.Typically, a liquid vaccine or vaccine component is freeze dried, oftenin the presence of an anti-caking agent (such as, for example, sugarssuch as sucrose or lactose). In some embodiments, the anti-caking agentis present, for example, at an initial concentration of 10-200 mg/mL.Lyophilization typically occurs over a series of steps, for instance acycle starting at −69° C., gradually adjusting to −24° C. over 3 hours,then retaining this temperature for 18 hours, then gradually adjustingto −16° C. over 1 hour, then retaining this temperature for 6 hours,then gradually adjusting to +34° C. over 3 hours, and finally retainingthis temperature over 9 hours.

Vaccines or vaccine components for use in accordance with the presentinvention may be incorporated into liposomes, cochleates, biodegradablepolymers such as poly-lactide, poly-glycolide andpoly-lactide-co-glycolides, or ISCOMS (immunostimulating complexes).

In certain situations, it may be desirable to prolong the effect of avaccine or for use in accordance with the present invention, for exampleby slowing the absorption of one or more vaccine components. Such delayof absorption may be accomplished, for example, by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the product then depends upon itsrate of dissolution, which in turn, may depend upon size and form.Alternatively or additionally, delayed absorption may be accomplished bydissolving or suspending one or more vaccine components in an oilvehicle. Injectable depot forms can also be employed to delayabsorption. Such depot forms can be prepared by forming microcapsulematrices of one or more vaccine components a biodegradable polymersnetwork. Depending upon the ratio of polymer to vaccine component, andthe nature of the particular polymer(s) employed, the rate of releasecan be controlled.

Examples of biodegradable polymers that can be employed in accordancewith the present invention include, for example, poly(orthoesters) andpoly(anhydrides). One particular exemplary polymer ispolylactide-polyglycolide.

Depot injectable formulations may also be prepared by entrapping theproduct in liposomes or microemulsions, which are compatible with bodytissues.

Polymeric delivery systems can also be employed in non-depotformulations including, for example, oral formulations. For example,biodegradable, biocompatible polymers such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid, etc., can be used in oral formulations. Polysaccharideantigens or conjugates may be formulated with such polymers, for exampleto prepare particles, microparticles, extrudates, solid dispersions,admixtures, or other combinations in order to facilitate preparation ofuseful formulations (e.g., oral).

Additional Vaccine Components and Combinations

Vaccines for use in accordance with the present invention includepneumococcal polysaccharides (and/or conjugates), and may additionallyinclude one or more additional active agents (i.e., agents that exert abiological effect—not inert ingredients). It will be appreciated thatsuch additional agents may be formulated together with one or more othervaccine components, or may be maintained separately and combined at ornear the time of administration. In some embodiments, such additionalcomponents may be administered separately from some or all of the othervaccine components, within an appropriate time window for the relevanteffect to be achieved.

For example, it is common in vaccine preparation to include one or moreadjuvants. Adjuvants, generally, are agents that enhance the immuneresponse to an antigen. In some embodiments, adjuvants that enhance aTh1-type immune response are utilized.

To give but a few examples, adjuvants that are suitable for use inaccordance with the present invention include, but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example,

-   -   (a) MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene,        0.5% Tween 80, and 0.5% Span 85 (optionally containing various        amounts of MTP-PE (see below, although not required)) formulated        into submicron particles using a microfluidizer such as Model        110Y microfluidizer (Microfluidics, Newton, Mass.),    -   (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5%        pluronic-blocked polymer L121, and thr-MDP (see below) either        microfluidized into a submicron emulsion or vortexed to generate        a larger particle size emulsion, and    -   (c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, Mont.)        containing 2% Squalene, 0.2% Tween 80, and one or more bacterial        cell wall components from the group consisting of 3-O-deaylated        monophosphorylipid A (MPL™) described in U.S. Pat. No. 4,912,094        (Corixa), trehalose dimycolate (TDM), and cell wall skeleton        (CWS), preferably MPL+CWS (Detox™);

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (U.S. Pat. No. 5,057,540) may be used or particlesgenerated therefrom such as ISCOMs (immuno stimulating complexes);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also know as 529 (formerly known as RC529), which is formulatedas an aqueous form or as a stable emulsion, synthetic polynucleotidessuch as oligonucleotides containing CpG motif(s) (U.S. Pat. No.6,207,646);

(5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc.;

(6) detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT) either in a wild-type or mutant form, for example,where the glutamic acid at amino acid position 29 is replaced by anotheramino acid, preferably a histidine, in accordance with publishedinternational patent application number WO 00/18434 (see also WO02/098368 and WO 02/098369), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129(see, e.g., WO 93/13302 and WO 92/19265); and

(7) other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

Vaccines for use in accordance with the present invention may includeone or more bacterial toxins and/or their attenuated derivatives (i.e.,not in the form of a polysaccharide conjugate). The compositions of thisinvention may also include one or more proteins from Streptococcuspneumoniae. Examples of Streptococcus pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationWO02/083855, as well as that described in International PatentApplication WO02/053761. Alternatively or additionally, such vaccinesmay include one or more other bacterial antigens such as, for example,PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128,Sp130 and Sp133, and combinations thereof.

Vaccines for use in accordance with the present invention may include,or be administered concurrently with, other antimicrobial therapy. Forexample, such vaccines may include or be administered with one or moreagents that kills or retards growth of S. pneumoniae. Such agentsinclude, for example, penicillin, vancomycin, erythromycin,azithromycin, and clarithromycin, cefotaxime, ceftriaxone, levoflaxin,gatifloxacin.

Alternatively or additionally, vaccines for use in accordance with thepresent invention may include, or be administered with, one or moreother vaccines or therapies. For example, one or more non-pneumococcalantigens may be included in or administered with the vaccines.

The compositions of this invention may further include one or moreadditional antigens for use against otitis media caused by infectionwith other bacteria. Such bacteria include nontypable Haemophilusinfluenza, Moraxella catarrhalis (formerly known as Branhamellacatarrhalis) and Alloiococcus otitidis.

Examples of nontypable Haemophilus influenzae antigens suitable forinclusion include the P4 protein, also known as protein “e” (U.S. Pat.No. 5,601,831; International Patent Application WO03/078453), the P6protein, also known as the PAL or the PBOMP-1 protein (U.S. Pat. No.5,110,908; International Patent Application WO0100790), the P5 protein(U.S. Reissue patent No. 37, 741), the Haemophilus adhesion andpenetration protein (U.S. Pat. Nos. 6,245,337 and 6,676,948), the LKPtip adhesin protein (U.S. Pat. No. 5,643,725) and the NucA protein (U.S.Pat. No. 6,221,365).

Examples of Moraxella catarrhalis antigens suitable for inclusioninclude the UspA2 protein (U.S. Pat. Nos. 5,552,146, 6,310,190), the CDprotein (U.S. Pat. No. 5,725,862), the E protein (U.S. Pat. No.5,948,412) and the 74 kilodalton outer membrane protein (U.S. Pat. No.6,899,885).

Examples of Alloiococcus otitidis antigens suitable for inclusioninclude those identified in International Patent ApplicationWO03/048304.

The compositions of this invention may further include one or moreproteins from Neisseria meningitidis type B. Examples of Neisseriameningitidis type B proteins suitable for inclusion include thoseidentified in International Patent Applications WO03/063766,WO2004/094596, WO01/85772, WO02/16612 and WO01/87939.

Further additional antigens may comprise antigens of other infectiousdiseases such as, but not limited to, hepatitis A, hepatitis B,influenza, meningitis, polio virus, tetanus, varicella, diphtheria,measles, mumps, and rubella. Certain exemplary antigens include, but arenot limited to Hepatitis B surface antigen (HBsAg), Moraxellacatarrhalis outer membrane proteins, non-typable Haemophilus influenzaeproteins, N. meningitidis B outer membrane proteins. Other combinationscontemplated are the pneumococcal PS & protein of the invention incombination with viral antigens, for example, from influenza(attenuated, split, or subunit [e.g., surface glycoproteinsneuraminidase (NA) and haemagglutinin (HA). See, e.g., Chaloupka I. etal, Eur. Journal Clin. Microbiol. Infect. Dis. 15:121-127, 1996], RSV(e.g., F and G antigens or F/G fusions, see, eg, Schmidt A. C. et al, JVirol, p 4594-4603, 2001), and PIV3 (e.g., HN and F proteins, seeSchmidt et al. supra).

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The representativeexamples contain information, exemplification and guidance, which can beadapted to the practice of this invention in its various embodiments andthe equivalents thereof. It should be understood that these examples arefor illustrative purposes only and are not to be construed as limitingthis invention in any manner

EXEMPLIFICATION Example 1 Dose Ranging Study in NaïVe Adults 70 Years

A dose-ranging study was carried out which compared the safety,tolerability and immunogenicity of pneumococcal conjugate vaccine atthree dosage levels with pneumococcal polysaccharide unconjugatedvaccine in ambulatory elderly adults aged 70 years and older Immuneresponse to a single injection was measured for each of three doselevels of 7vPnC vaccine (PREVNAR®, pneumococcal 7-valent conjugatevaccine (Diphtheria CRM₁₉₇ protein), Wyeth Pharmaceuticals, Inc.,Philadelphia, Pa.), relative to the response to 23vPS vaccine(Pneumovax® 23, pneumococcal polysaccharide vaccine, Aventis PasteurMSD, Ltd., Berkshire, UK). Safety and tolerability of a single injectionof one of three dose levels of 7vPnC vaccine relative to that of 23vPSvaccine were also assessed.

Study Design

A total of 443 individuals, 70 years and older without prior 23vPSvaccination were enrolled in a randomized, open-label, and controlled,study. Three different dosage levels of pneumococcal conjugate vaccinerepresenting 1×, 2×, and 4× the 7vPnC dosage were evaluated. Because nohigher dosage for PREVNAR® 7vPnC formulations were available, the 2×dosage was approximated by mixing one vial of PREVNAR® 7vPnC at 2μg/serotype (4 μg for serotype 6B) with one vial of lyophilized 9valentPnC vaccine (9vPnC) at 2 μg/serotype (containing additional serotypes 1and 5). The 4× dosage was obtained by mixing two vials of PREVNAR® 7vPnCwith 2 vials of 9-valent vaccine. Individuals were randomized in a1:1:1:1 ratio to receive a single injection of one of the threeconjugate formulations or a single injection of 23vPS.

Table 1 below outlines the dosing plan.

TABLE 1 Dosing Plan for Adult Pneumococcal Dose Ranging Study Year 2Dose 2 Year 1 -- Dose 1 (12 months after Dose 1) Group N Vaccine Group NVaccine 1 100 1x 1a 50 23vPS PREVNAR ® 1b 50 1x PREVNAR ® 2 100 1x 2a 5023vPS PREVNAR ® + 2b 50 1x 1x 9vPnC PREVNAR ® + 1x 9vPnC 3 100 2x 3a 5023vPS PREVNAR ® + 3b 50 2x 2x 9vPnC PREVNAR ® + 2x 9vPnC 4 100 23vPS 4 100 PREVNAR ®

Table 2 outlines the vaccines administered for the studies in thisExample, as well as the second year Dose 2 studies described in Example2.

TABLE 2 Vaccines administered in the Dose-ranging study Total Dose/Polysaccha- CRM197 Vol- serotype ride Dose dose Alum ume Vaccines (μg)(μg) (μg) (mg) (ml) PREVNAR ® 2 (6B = 16 18 0.125 0.5 4 μg) 1 x 4 (6B =40 37 0.125 0.5 PREVNAR ® + 8 μg) 1 x 9vPnC 2 x 8 (6B = 80 74 0.250 1.0PREVNAR ® + 16 μg)  2 x 9vPnC 23vPS 25 575 — — 0.5Results from Single Dose of Vaccine.

A total of 443 subjects, with approximately 110 subjects per treatmentgroup were vaccinated with PnC 2 μg/serotype (PREVNAR®, 7vPnC), 4 μgPnC, 8 μg PnC, or 23vPS (PNEUMOVAX®23). The groups were well balancedfor demographics and pre-immunization antibody levels. Immune responsesto vaccine were assessed by ELISA analyses to measure serotype specificantibody levels; and opsonophagocytosis (OPA) analyses to determineopsonic titer activity levels. Local and systemic reactions wereassessed and recorded up to two weeks post vaccination dosing.

ELISA results for 7 serotypes following a single vaccine dose are shownin Table 3. The ELISA geometric mean antibody concentration (GMCs) for7vPnC were superior to 23vPS for all but one (serotype 19F) of theserotypes assessed, demonstrating increased levels of mean antibodyconcentration for the conjugate vaccine treatment group as compared tothe unconjugated polysaccharide vaccine treatment group, for six of theseven serotypes assessed.

Initial OPA results for serotypes 6B, 9V, 18C, 19F and 23F, following asingle dose of vaccine are shown in Table 4. The OPA geometric meantiters (GMTs) for 7vPnC were superior to 23vPS for serotypes 9V, 18C,23F, demonstrating increased activity against three of five serotypesassessed.

For all responses and reactions recorded, pain at the injection site andredness were the most frequently observed local reactions after dose 1.Pain at the injection site and redness were comparable between the 7vPnCand 23vPS groups with a trend toward higher responses in the 7vPnCgroup. See Table 5 and Table 6 for a summary of the safety andtolerability assessment results following a single dose of vaccine.Safety and tolerability between 7vPnC and 23vPS were comparable with atrend toward higher responses in the 7vPnC group, as the 4 μg dose wasalso comparable to the 23vPS, while the 8 mg dose produced higher localreactogenicity. The incidence of injection site events and systemicreactions were acceptable for all groups tested.

TABLE 3 Post Dose 1 Pneumococcal ELISA Geometric Mean AntibodyConcentrations (μg/mL) (Dose 1 All-Available Immunogenicity Population)Randomized Sampling Time^(a) Treatment 1 Month Post Dose 1 SerotypeGroup N^(b) GMC^(c) 95% CI^(d)  4 7vPnC 110 3.1 (2.2, 4.3) 23vPS 107 1.4(1.1, 2.0)  6B 7vPnC 110 8.0  (6.0, 10.8) 23vPS 107 4.4 (3.4, 5.8)  9V7vPnC 110 9.8  (7.5, 12.8) 23vPS 107 3.6 (2.8, 4.6) 14 7vPnC 110 17.1(12.3, 24.0) 23vPS 107 8.5  (6.0, 12.1) 18C 7vPnC 110 13.0 (10.1, 16.7)23vPS 107 6.8 (5.2, 8.9) 19F 7vPnC 110 5.5 (4.1, 7.4) 23vPS 107 4.4(3.4, 5.8) 23F 7vPnC 110 12.4  (9.0, 17.0) 23vPS 107 3.8 (2.9, 5.0)^(a)Protocol specified timing for blood sample. ^(b)N = the number ofsubjects with assay results for the specified serotype and the givenvisit. ^(c)Geometric Mean Concentrations (GMCs) were calculated usingall subjects with available data for the specified blood draw.^(d)Confidence limits (CI) are back transforms of a confidence intervalbased on Student's t-distribution for the mean logarithm of theconcentrations.

TABLE 4 Pneumococcal OPA Geometric Mean Antibody Titers for Dose 1 (Dose1 Evaluable Immunogenicity Population) Randomized Sampling Time^(a)Sero- Treatment Pre-Dose 1 1 Month Post-Dose 1 Fold Rise (Post/Pre) typeGroup N^(b) GMT^(c) 95% CI^(d) N^(b) GMT^(c) 95% CI^(d) N^(b) GMFR^(e)95% CI^(d)  1 7vPnC 99 22.7 (16.9, 30.6) 99 23.4 (17.2, 31.8) 99 1.0(0.8, 1.4) 2x 107 24.9 (18.0, 34.4) 108 306.4 (214.9, 436.9) 107 12.6 (8.5, 18.6) 4x 107 23.1 (17.2, 31.2) 107 438.3 (318.3, 603.5) 107 18.9(13.2, 27.1) 23vPS 100 28.1 (20.4, 38.6) 100 147.0 (103.8, 208.4) 1005.2 (3.5, 7.8)  4 7vPnC 99 12.9  (9.0, 18.4) 99 1504.9 (1025.0, 2209.6)99 116.9  (71.5, 190.9) 2x 107 15.2 (10.7, 21.6) 108 1505.0 (1015.2,2231.1) 107 98.1  (57.5, 167.4) 4x 107 15.4 (10.7, 22.1) 107 2021.6(1490.5, 2742.1) 107 131.4  (82.9, 208.2) 23vPS 100 14.1  (9.8, 20.3)100 661.7 (447.9, 977.6) 100 46.9 (29.2, 75.3)  5 7vPnC 99 20.2 (14.3,28.5) 99 17.9 (13.3, 24.0) 99 0.9 (0.7, 1.2) 2x 107 21.0 (15.0, 29.5)108 278.3 (182.1, 425.2) 107 13.2  (8.5, 20.3) 4x 107 19.2 (14.0, 26.4)107 444.0 (286.3, 688.4) 107 23.1 (15.4, 34.7) 23vPS 100 21.7 (16.0,29.4) 100 250.7 (171.0, 367.7) 100 11.6  (7.5, 17.8)  6B 7vPnC 96 64.0 (39.0, 105.0) 99 1526.2 (1043.8, 2231.6) 96 22.6 (13.8, 37.0) 2x 10074.0  (45.1, 121.5) 107 2536.1 (1760.3, 3653.9) 99 34.1 (20.1, 57.9) 4x98 52.9 (32.9, 84.9) 107 3914.4 (2929.5, 5230.5) 98 76.9 (47.4, 124.8)23vPS 93 57.7 (34.2, 97.3) 99 830.0  (553.3, 1245.0) 92 13.7  (8.0,23.2)  9V 7vPnC 99 116.9  (76.1, 179.5) 99 3052.5 (2193.3, 4248.1) 9926.1 (15.3, 44.7) 2x 105 172.3 (109.1, 272.1) 108 3511.3 (2547.1,4840.3) 105 21.1 (13.3, 33.6) 4x 107 147.6  (97.1, 224.4) 107 5239.2(3976.8, 6902.4) 107 35.5 (21.9, 57.6) 23vPS 99 139.2  (88.3, 219.4) 99981.9  (697.5, 1382.2) 98 6.9  (4.4, 10.9) 14 7vPnC 99 110.5  (63.0,193.7) 99 2526.7 (1640.4, 3891.9) 99 22.9 (12.7, 41.3) 2x 107 112.4 (67.6, 187.0) 108 2547.4 (1699.3, 3818.9) 107 22.3 (12.9, 38.6) 4x 10768.3  (41.0, 113.8) 106 3433.1 (2569.9, 4586.1) 106 51.6 (28.6, 93.0)23vPS 100 93.7  (55.6, 158.0) 99 1024.0  (628.7, 1667.8) 99 11.1  (6.4,19.2) 18C 7vPnC 99 47.4 (32.3, 69.4) 99 1364.5  (937.6, 1985.8) 99 28.8(18.4, 45.0) 2x 107 50.7 (35.3, 72.9) 108 1711.1 (1159.6, 2525.1) 10733.9 (21.3, 54.1) 4x 107 35.3 (25.5, 48.8) 106 2172.1 (1497.1, 3151.6)106 61.9 (41.6, 92.1) 23vPS 100 42.8 (29.5, 62.1) 100 458.3 (302.7,693.7) 100 10.7  (7.3, 15.6) 19F 7vPnC 98 15.1 (10.8, 21.1) 99 200.4(130.3, 308.2) 98 13.4  (8.4, 21.4) 2x 106 14.5 (10.4, 20.2) 108 148.4 (96.9, 227.1) 106 10.5  (6.9, 16.2) 4x 107 17.4 (12.6, 24.1) 106 359.7(246.9, 524.0) 106 20.4 (14.3, 29.1) 23vPS 100 12.2  (8.9, 16.8) 100203.7 (134.1, 309.3) 100 16.7 (10.8, 25.7) 23F 7vPnC 96 40.9 (26.1,64.0) 99 1403.2  (898.6, 2191.2) 96 35.7 (20.6, 61.8) 2x 105 55.7 (35.4,87.7) 106 1205.9  (822.8, 1767.3) 103 20.8 (12.9, 33.6) 4x 104 46.2(30.3, 70.3) 105 1929.9 (1370.1, 2718.3) 102 39.2 (24.9, 61.9) 23vPS 9944.8 (28.7, 69.9) 97 291.1 (193.9, 437.1) 96 6.1 (4.2, 8.8) ^(a)Protocolspecified timing for blood sample. ^(b)N = Number of subjects with assayresults for the specified serotype and the given visit. ^(c)GeometricMean Titers (GMTs) were calculated using all subjects with availabledata for the specified blood draw. ^(d)Confidence limits (CI) are backtransforms of a confidence interval based on Student's t-distributionfor the mean logarithm of the concentrations or fold-rises.^(e)Geometric Mean Fold Rises (GMFRs) were calculated using all subjectswith available data from both the pre-dose and post-dose blood draws. 2x= 9vPnC reconstituted with 7vPnC. 4x = 2 doses 9vPnC reconstituted with2 doses 7vPnC.

TABLE 5 Number (%) of Subjects Reporting Injection Site Events Within 7Days Following Dose 1 Actual Treatment Group 7vPnC 2x 4x 23vPS LocalReaction N^(a) n^(b) % N^(a) n^(b) % N^(a) n^(b) % N^(a) n^(b) %p-value^(c) Redness Any 103 35 34.0 102 25 24.5 105 51 48.6 100 23 23.00.090 Significant^(d) 99 12 12.1 102 12 11.8 101 34 33.7 99 9 9.10.645 >7 cm^(e) 99 2 2.0 100 0 0.0 100 7 7.0 99 2 2.0 >0.999 SwellingAny 102 20 19.6 100 16 16.0 106 44 41.5 99 17 17.2 0.718 Significant^(d)99 5 5.1 100 6 6.0 98 20 20.4 97 5 5.2 >0.999 >7 cm^(e) 99 0 0.0 98 00.0 97 3 3.1 97 1 1.0 0.495 Pain at injection site Any 104 40 38.5 10634 32.1 109 58 53.2 104 26 25.0 0.052 Significant^(f) 99 4 4.0 102 2 2.0108 14 13.0 103 4 3.9 >0.999 Any Injection Site Reaction^(g) 106 57 53.8105 46 43.8 108 82 75.9 100 43 43.0 0.128 ^(a)N represents the number ofsubjects with known values. ^(b)n represents the number of subjects withthe specified event. ^(c)Fisher Exact test, two-sided, for percent ofsubjects between the 7vPnC and 23vPS groups. ^(d)Significant is definedas having a diameter for the involved area >8 caliper units or 4.0 cm,including subjects with local reactions >7.0 cm. ^(e)Subjects with localreactions >7.0 cm were encouraged not to return. ^(f)Significant isdefined as interfering with limb movement. ^(g)Any injection site eventincludes any pain, any swelling, and any redness. Subjects were includedin this category if they experienced an event or had ‘no’ for all daysfor all events (i.e., any missing value excludes the subject unless theyexperienced an event). 2x = 9vPnC reconstituted with 7vPnC. 4x = 2 doses9vPnC reconstituted with 2 doses 7vPnC.

TABLE 6 Number (%) of Subjects Reporting Systemic Reactions Within 7Days Following Dose 1 Actual Treatment Group 7vPnC 2x 4x 23vPS SystemicReaction N^(a) n^(b) % N^(a) n^(b) % N^(a) n^(b) % N^(a) n^(b) %p-value^(c) Fever ≧38° C. 101 1 1.0 106 2 1.9 110 3 2.7 104 3 2.9 0.621Fever >39° C. 101 0 0.0 106 0 0.0 110 0 0.0 104 0 0.0 >0.999 Fever >40°C. 101 0 0.0 106 0 0.0 110 0 0.0 104 0 0.0 >0.999 Fatigue 106 24 22.6108 23 21.3 112 23 20.5 105 22 21.0 0.868 Headache 104 11 10.6 108 1513.9 112 17 15.2 106 14 13.2 0.671 Chills 105 3 2.9 107 4 3.7 112 9 8.0105 9 8.6 0.134 Rash 105 2 1.9 107 3 2.8 109 7 6.4 105 2 1.9 >0.999Vomiting 104 0 0.0 107 2 1.9 111 1 0.9 104 2 1.9 0.498 Decreasedappetite 106 5 4.7 107 9 8.4 112 7 6.3 105 9 8.6 0.284 Muscle pain 10520 19.0 103 13 12.6 111 26 23.4 106 14 13.2 0.267 Joint pain 103 8 7.8106 12 11.3 108 12 11.1 101 6 5.9 0.783 Medication to treat 95 1 1.1 1001 1.0 101 2 2.0 91 1 1.1 >0.999 fever Any new medications 93 3 3.2 98 44.1 101 2 2.0 93 4 4.3 >0.999 Any Systemic 101 40 39.6 105 40 38.1 10646 43.4 102 39 38.2 0.886 Reaction^(d) ^(a)N represents the number ofsubjects with known values. ^(b)n represents the number of subjects withthe specified event. ^(c)Fisher Exact test, two-sided, for percent ofsubjects between the 7vPnC and 23cPS groups. ^(d)Any systemic reactionexcludes any new medications and any medications to treat a fever.Subjects were included in this category if they experienced an event orhad ‘no’ for all days for all events (i.e., any missing value excludesthe subject unless they experienced an event). 2x = 9vPnC reconstitutedwith 7vPnC; 4x = 2 doses 9vPnC reconstituted with 2 doses 7vPnC.

Summary of First Dose Study Results

As discussed above, the benefits of a conjugate vaccine in naïve adultscompared to unconjugated polysaccharide vaccine include demonstratedhigher antibody levels (ELISA) and better functional antibody activity(OPA) responses. Both could be demonstrated after a single dose, with anacceptable safety profile.

Example 2 Dose Ranging Study—Second Dosing Study Design

See Study Design description in Example 1 above, and Table 1 and Table 2for description of study, dosing regimen and formulations used in year 2second dosing of this study. Twelve months after the first dose ofpneumococcal vaccine, individuals in each conjugate cohort werere-randomized to receive either the same formulation as the first doseor 23vPS to assess the response to subsequent conjugate orpolysaccharide vaccine. Individuals who initially received 23vPSreceived a dose of 7-valent pneumococcal conjugate vaccine (PREVNAR®). Atotal of 314 subjects (out of 443) participated in the second year ofthe study.

Results after a Second Dose of Vaccine

Immune responses as well as safety and tolerability to a pneumococcalconjugate dose after an initial dose of either PnC or 23vPS when given12 months after a first injection were assessed. Additionally, immuneresponses as well as safety and tolerability of 7vPnC and/or 23vPS whengiven after a prior dose of conjugate vaccine, were assessed. As inExample 1, blood samples were obtained from subjects prior to and onemonth post vaccination. Serotype specific antibody production andfunctional antibody responses were measured by ELISA and OPA analyses,respectively. Local and systemic reactions were assessed and recorded upto two weeks post vaccination dosing.

ELISA geometric mean antibody concentration (GMCs) results for each of 7common serotypes following first and second vaccine doses are shown inTables 7, 8, 9, and 10. The ELISA results for comparative treatmentgroups are shown in each of Table 7 (23vPS Dose 1 vs. 7vPnC Dose 1/23vPSDose 2); Table 8 (7vPnC Dose 1 vs. 7vPnC Dose 1/7vPnC Dose 2); and Table9 (7vPnC Dose 1 vs. 23vPS Dose 1/7vPnC Dose 2). Table 10 shows detailedimmunogenicity results for each of the treatment groups.

TABLE 7 Pneumococcal ELISA Geometric Mean Antibody Concentrations(μg/mL) (Dose 1 and Dose 2 Evaluable Immunogenicity Populations)Sampling Time^(a) Sero- 23vPS, Post-Dose 1 7vPnC/23vPS, Post-Dose 2 typeN^(b) GMC^(c) 95% CI^(d) N^(b) GMC^(c) 95% CI^(d)  4 62 1.6 (1.1, 2.5)30 1.5 (0.9, 2.6)   6B 62 4.4 (3.2, 6.0) 30 5.0 (2.7, 9.1)   9V 62 3.4(2.5, 4.6) 30 6.1 (3.6, 10.3) 14 62 8.9  (5.3, 15.0) 30 15.0 (8.4, 26.8)18C 62 6.6 (4.6, 9.6) 30 7.1 (4.6, 10.9) 19F 62 4.5 (3.1, 6.6) 30 8.0(4.7, 13.8) 23F 62 3.8 (2.6, 5.6) 30 7.5 (3.7, 15.1) ^(a)Protocolspecified timing for blood sample. ^(b)N = the number of subjects withassay results for the specified serotype and the given visit.^(c)Geometric Mean Concentrations (GMCs) were calculated using allsubjects with available data for the specified blood draw.^(d)Confidence limits (CI) are back transforms of a confidence intervalbased on Student's t-distribution for the mean logarithm of theconcentrations.

TABLE 8 Pneumococcal ELISA Geometric Mean Antibody Concentrations(μg/mL) (Dose 1 and Dose 2 Evaluable Immunogenicity Populations)Sampling Time^(a) Sero- 7vPnC, Post-Dose 1 7vPnC/7vPnC, Post-Dose 2 typeN^(b) GMC^(c) 95% CI^(d) N^(b) GMC^(c) 95% CI^(d)  4 61 2.6 (1.7, 4.0) 31 3.3 (1.9, 5.7)  6B 61 7.4 (4.7, 11.8) 31 8.3  (4.1, 16.8)  9V 61 8.5(6.2, 11.8) 31 6.6 (4.5, 9.5) 14 61 16.1 (9.9, 26.1) 31 17.6 (10.2,30.3) 18C 61 11.4 (8.4, 15.5) 31 10.0  (7.2, 13.8) 19F 61 5.6 (3.9,8.2)  31 6.7  (3.9, 11.3) 23F 61 12.5 (8.1, 19.4) 31 16.5 (11.5, 23.7)^(a)Protocol specified timing for blood sample. ^(b)N = the number ofsubjects with assay results for the specified serotype and the givenvisit. ^(c)Geometric Mean Concentrations (GMCs) were calculated usingall subjects with available data for the specified blood draw.^(d)Confidence limits (CI) are back transforms of a confidence intervalbased on Student's t-distribution for the mean logarithm of theconcentrations.

Treatment group 7vPnC/23vPS: The point estimates for ELISA GMCs for asubsequent dose of 23vPS following 7vPnC were higher than after aninitial dose of 23vPS alone for all but one (serotype 4) of the commonserotypes. See Table 7.

Treatment group 7vPnC/7vPnC: The point estimates for ELISA GMCs after asecond dose of 7vPnC were higher than after an initial dose of 23vPS.The ELISA GMC's for a subsequent dose of 7vPnC following 7vPnC weresimilar to an initial dose of 7vPnC. See Table 3 above for the 23vPSresults as comparison. See Table 8.

TABLE 9 Pneumococcal ELISA Geometric Mean Antibody Concentrations(μg/mL) (Dose 1 and Dose 2 Evaluable Immunogenicity Populations)Sampling Time^(a) Sero- 7vPnC, Post-Dose 1 23vPS/7vPnC, Post-Dose 2 typeN^(b) GMC^(c) 95% CI^(d) N^(b) GMC^(c) 95% CI^(d)  4 61 2.6 (1.7, 4.0) 62 1.0 (0.6, 1.5)  6B 61 7.4 (4.7, 11.8) 62 2.7 (1.9, 4.0)  9V 61 8.5(6.2, 11.8) 62 2.8 (2.0, 3.9) 14 61 16.1 (9.9, 26.1) 62 6.9  (4.2, 11.1)18C 61 11.4 (8.4, 15.5) 62 5.2 (3.8, 7.3) 19F 61 5.6 (3.9, 8.2)  62 2.2(1.5, 3.3) 23F 61 12.5 (8.1, 19.4) 62 3.7 (2.3, 6.2) ^(a)Protocolspecified timing for blood sample. ^(b)N = the number of subjects withassay results for the specified serotype and the given visit.^(c)Geometric Mean Concentrations (GMCs) were calculated using allsubjects with available data for the specified blood draw.^(d)Confidence limits (CI) are back transforms of a confidence intervalbased on Student's t-distribution for the mean logarithm of theconcentrations.

Treatment group 23vPnC/7vPnC: The point estimates for ELISA GMCs for asubsequent dose of 7vPnC following 23vPS were lower than after aninitial dose of 7vPnC for all serotypes and are statistically inferiorfor 6 of the 7 serotypes. These data showed induction ofhyporesponsiveness after a single dose of 23vPS vaccine. See Table 9.

Functional antibody response as measured by OPA analyses for each of theseven common serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, following asingle dose of vaccine or a second dose of vaccine are shown in Table11.

TABLE 10 Pneumococcal ELISA Geometric Mean Antibody Concentrations(μg/mL) (Dose 1 and Dose 2 Evaluable Immunogenicity Populations)Randomized Treatment Sampling Time^(a) Sero- (Dose 1/Dose Pre-Dose 1Post-Dose 1 Pre-Dose 2 Post-Dose 2 type 2) N^(b) GMC^(c) 95% CI^(d)N^(b) GMC^(c) 95% CI^(d) N^(b) GMC^(c) 95% CI^(d) N^(b) GMC^(c) 95%CI^(d)  4 7vPnC 61 0.2 (0.1, 0.3) 61 2.6 (1.7, 4.0) 23vPS 62 0.2 (0.1,0.3) 62 1.6 (1.1, 2.5) 7vPnC/23vPS 30 0.7 (0.4, 1.2) 30 1.5 (0.9, 2.6)7vPnC/7vPnC 31 0.9 (0.4, 1.7) 31 3.3 (1.9, 5.7) 23vPS/7vPnC 62 0.5 (0.3,0.8) 62 1.0 (0.6, 1.5)  6B 7vPnC 61 1.1 (0.8, 1.7) 61 7.4  (4.7, 11.8)23vPS 62 1.0 (0.7, 1.4) 62 4.4 (3.2, 6.0) 7vPnC/23vPS 30 2.0 (1.1, 3.7)30 5.0 (2.7, 9.1) 7vPnC/7vPnC 31 2.3 (1.2, 4.5) 31 8.3  (4.1, 16.8)23vPS/7vPnC 62 1.5 (1.0, 2.1) 62 2.7 (1.9, 4.0)  9V 7vPnC 61 0.9 (0.7,1.3) 61 8.5  (6.2, 11.8) 23vPS 62 0.7 (0.5, 1.0) 62 3.4 (2.5, 4.6)7vPnC/23vPS 30 3.9 (2.3, 6.7) 30 6.1  (3.6, 10.3) 7vPnC/7vPnC 31 3.3(2.0, 5.4) 31 6.6 (4.5, 9.5) 23vPS/7vPnC 62 1.4 (1.0, 2.0) 62 2.8 (2.0,3.9) 14 7vPnC 61 2.8 (1.7, 4.5) 61 16.1  (9.9, 26.1) 23vPS 62 1.6 (1.0,2.4) 62 8.9  (5.3, 15.0) 7vPnC/23vPS 30 7.8  (3.8, 16.4) 30 15.0  (8.4,26.8) 7vPnC/7vPnC 31 10.5  (5.1, 21.7) 31 17.6 (10.2, 30.3) 23vPS/7vPnC62 5.5 (3.3, 9.2) 62 6.9  (4.2, 11.1) 18C 7vPnC 61 1.2 (0.9, 1.6) 6111.4  (8.4, 15.5) 23vPS 62 1.0 (0.7, 1.4) 62 6.6 (4.6, 9.6) 7vPnC/23vPS30 5.0 (3.0, 8.5) 30 7.1  (4.6, 10.9) 7vPnC/7vPnC 31 5.6 (3.6, 8.6) 3110.0  (7.2, 13.8) 23vPS/7vPnC 62 3.2 (2.2, 4.7) 62 5.2 (3.8, 7.3) 19F7vPnC 61 1.4 (0.9, 2.0) 61 5.6 (3.9, 8.2) 23vPS 62 1.1 (0.8, 1.5) 62 4.5(3.1, 6.6) 7vPnC/23vPS 30 2.8 (1.6, 4.9) 30 8.0  (4.7, 13.8) 7vPnC/7vPnC31 1.6 (0.9, 3.0) 31 6.7  (3.9, 11.3) 23vPS/7vPnC 62 1.5 (1.0, 2.3) 622.2 (1.5, 3.3) 23F 7vPnC 61 1.1 (0.8, 1.5) 61 12.5  (8.1, 19.4) 23vPS 620.9 (0.6, 1.3) 62 3.8 (2.6, 5.6) 7vPnC/23vPS 30 6.1  (2.8, 13.1) 30 7.5 (3.7, 15.1) 7vPnC/7vPnC 31 3.8 (2.1, 6.6) 31 16.5 (11.5, 23.7)23vPS/7vPnC 62 1.4 (0.9, 2.1) 62 3.7 (2.3, 6.2) ^(a)Protocol specifiedtiming for blood sample. ^(b)N = the number of subjects with assayresults for the specified serotype and the given visit. ^(c)GeometricMean Concentrations (GMCs) were calculated using all subjects withavailable data for the specified blood draw. ^(d)Confidence limits (CI)are back transforms of a confidence interval based on Student'st-distribution for the mean logarithm of the concentrations.

TABLE 11 Pneumococcal OPA Geometric Mean Antibody Titers for Dose 2(Evaluable Immunogenicity Population) Randomized Sampling Time^(a)Treatment Group Pre-Dose 2 Month Post-Dose 2 Fold Rise (Post/Pre)Serotype (Dose 1/Dose 2) N^(b) GMT^(c) 95% CI^(d) N^(b) GMT^(c) 95%CI^(d) N^(b) GMFR^(e) 95% CI^(d)  1 7vPnC/23vPS 30 5.2 (4.1, 6.6)  3090.5 (46.4, 176.6) 30 17.5  (9.4, 32.9) 7vPnC/7vPnC 33 7.2 (5.1, 10.2)33 6.3 (4.5, 8.9)  33 0.9 (0.7, 1.2) 2x/23vPS 33 24.9 (14.9, 41.4)  3294.5 (51.8, 172.6) 32 4.0 (2.4, 6.7) 2x/2x 36 33.3 (18.0, 61.6)  36109.7 (58.9, 204.4) 36 3.3 (2.0, 5.5) 4x/23vPS 30 36.8 (19.4, 69.6)  3082.5 (48.9, 139.4) 30 2.2 (1.3, 3.8) 4x/4x 33 30.7 (17.2, 54.6)  33168.2 (90.2, 313.7) 33 5.5 (3.1, 9.7) 23vPS/7vPnC 63 16.4 (11.1, 24.2) 63 15.7 (10.6, 23.0)  63 1.0 (0.8, 1.2)  4 7vPnC/23vPS 30 330.1 (140.3,776.7)  30 1203.8 (646.4, 2241.7) 30 3.6 (1.9, 7.1) 7vPnC/7vPnC 33 397.9(185.7, 852.6)  33 1433.0 (935.7, 2194.7) 33 3.6 (1.8, 7.2) 2x/23vPS 33235.4 (107.7, 514.2)  32 1046.4 (636.1, 1721.3) 32 4.7 (2.5, 8.7) 2x/2x36 456.1 (238.1, 873.9)  36 1149.4 (653.4, 2021.9) 36 2.5 (1.6, 4.0)4x/23vPS 30 574.7 (321.4, 1027.5) 30 851.2 (465.3, 1557.0) 30 1.5 (1.1,2.1) 4x/4x 33 480.7 (276.2, 836.8)  33 1922.9 (1277.1, 2895.4)  33 4.0(2.5, 6.3) 23vPS/7vPnC 63 210.0 (128.5, 343.2)  63 506.4 (349.7, 733.4) 63 2.4 (1.7, 3.5)  5 7vPnC/23vPS 30 8.4 (5.1, 13.8) 30 150.5 (81.4,278.1) 30 18.0  (9.0, 35.9) 7vPnC/7vPnC 33 9.5 (5.1, 17.6) 33 7.5 (4.8,11.9) 33 0.8 (0.5, 1.2) 2x/23vPS 33 39.5 (19.6, 79.7)  32 162.4 (90.9,290.2) 32 4.5 (2.6, 7.6) 2x/2x 36 48.9 (25.2, 94.9)  36 130.5 (66.7,255.4) 36 2.7 (1.6, 4.4) 4x/23vPS 30 59.7 (25.7, 138.6) 30 189.6 (105.8,339.8)  30 3.2 (1.7, 5.9) 4x/4x 32 39.7 (18.3, 86.3)  33 261.4 (142.5,479.6)  32 6.3  (3.6, 11.0) 23vPS/7vPnC 62 55.3 (32.9, 93.0)  63 62.6(38.4, 102.1) 62 1.2 (0.9, 1.6)  6B 7vPnC/23vPS 30 157.6 (53.3, 465.7)30 1072.4 (460.2, 2499.2) 30 6.8  (3.2, 14.5) 7vPnC/7vPnC 33 284.3(110.2, 733.9)  33 2323.1 (1207.3, 4469.9)  33 8.2  (3.5, 18.9) 2x/23vPS33 461.0 (178.2, 1192.1) 33 941.5 (450.7, 1966.8) 33 2.0 (1.1, 3.7)2x/2x 36 261.0 (98.3, 693.0) 36 2298.8 (1045.5, 5054.4)  36 8.8  (3.8,20.5) 4x/23vPS 30 500.3 (186.6, 1341.2) 30 691.4 (308.7, 1548.3) 30 1.4(0.7, 2.7) 4x/4x 32 459.4 (179.9, 1173.2) 33 3052.5 (1743.8, 5343.3)  326.7  (2.9, 15.7) 23vPS/7vPnC 63 186.1 (104.0, 332.8)  63 438.9 (248.9,774.1)  63 2.4 (1.4, 4.0)  9V 7vPnC/23vPS 30 1047.9 (576.1, 1906.2) 302144.9 (1006.4, 4571.1)  30 2.0 (0.9, 4.9) 7vPnC/7vPnC 33 902.7 (434.7,1874.8) 33 2274.8 (1277.7, 4049.9)  33 2.5 (1.5, 4.4) 2x/23vPS 33 1045.7(538.8, 2029.7) 33 2323.1 (1139.3, 4736.8)  33 2.2 (1.2, 4.0) 2x/2x 36844.7 (420.7, 1696.0) 36 3128.2 (1704.6, 5740.7)  36 3.7 (2.1, 6.5)4x/23vPS 30 2001.2 (1312.7, 3050.8)  30 2702.4 (1385.5, 5270.8)  30 1.4(0.7, 2.6) 4x/4x 32 1191.7 (666.6, 2130.1) 33 4010.9 (2614.7, 6152.5) 32 3.4 (2.2, 5.1) 23vPS/7vPnC 63 463.7 (267.5, 803.9)  63 1290.2 (792.2,2101.2) 63 2.8 (1.8, 4.3) 14 7vPnC/23vPS 29 976.2 (415.5, 2293.5) 302521.4 (1308.4, 4859.0)  29 2.5 (1.3, 5.1) 7vPnC/7vPnC 33 1433.0 (730.8,2810.1) 33 3183.4 (1948.6, 5200.8)  33 2.2 (1.3, 3.8) 2x/23vPS 33 1137.4(649.6, 1991.6) 33 2048.0 (1150.8, 3644.7)  33 1.8 (1.0, 3.4) 2x/2x 351108.4 (549.3, 2236.6) 36 1689.3 (946.9, 3013.8) 35 1.5 (0.8, 2.7)4x/23vPS 30 1123.1 (583.7, 2161.2) 30 2001.2 (1234.7, 3243.7)  30 1.8(1.1, 3.0) 4x/4x 32 1357.0 (728.9, 2526.4) 33 2580.3 (1645.1, 4047.3) 32 1.9 (1.0, 3.6) 23vPS/7vPnC 63 652.2 (369.3, 1151.9) 63 1221.1 (781.2,1908.7) 63 1.9 (1.4, 2.5) 18C 7vPnC/23vPS 30 280.8 (126.5, 623.5)  30955.4 (549.2, 1662.1) 30 3.4 (2.0, 5.7) 7vPnC/7vPnC 33 397.9 (204.8,773.1)  33 1067.9 (648.4, 1758.9) 33 2.7 (1.7, 4.2) 2x/23vPS 33 203.2(95.1, 434.1) 33 593.1 (301.1, 1168.4) 33 2.9 (1.7, 5.0) 2x/2x 36 438.9(218.8, 880.3)  36 844.7 (480.0, 1486.3) 36 1.9 (1.2, 3.1) 4x/23vPS 30445.7 (243.2, 816.8)  30 645.1 (354.8, 1172.7) 30 1.4 (1.0, 2.1) 4x/4x32 534.7 (245.1, 1166.2) 33 1237.1 (637.8, 2399.6) 32 2.4 (1.3, 4.2)23vPS/7vPnC 63 196.6 (115.0, 336.1)  63 312.1 (190.2, 512.0)  63 1.6(1.2, 2.1) 19F 7vPnC/23vPS 30 71.8 (31.2, 165.2) 30 435.5 (244.0,777.5)  30 6.1  (2.5, 14.5) 7vPnC/7vPnC 33 37.9 (18.2, 78.7)  33 284.3(147.5, 548.2)  33 7.5  (4.3, 13.0) 2x/23vPS 33 60.1 (25.1, 144.0) 33261.4 (144.8, 472.1)  33 4.4 (2.2, 8.6) 2x/2x 36 52.8 (25.7, 108.6) 36237.0 (121.9, 461.0)  36 4.5 (2.3, 8.7) 4x/23vPS 30 50.8 (19.3, 133.9)30 406.4 (190.4, 867.1)  30 8.0  (3.9, 16.4) 4x/4x 32 64.0 (29.6, 138.2)33 250.7 (126.3, 497.6)  32 3.8 (1.9, 7.5) 23vPS/7vPnC 63 61.2 (32.8,114.4) 63 114.7 (65.4, 201.1) 63 1.9 (1.2, 2.9) 23F 7vPnC/23vPS 30 601.9(265.7, 1363.3) 30 1290.2 (612.1, 2719.3) 30 2.1 (1.2, 3.8) 7vPnC/7vPnC33 605.7 (235.2, 1559.9) 33 4948.3 (2650.2, 9239.4)  33 8.2  (3.8, 17.5)2x/23vPS 33 284.3 (112.4, 719.3)  33 631.7 (281.2, 1419.0) 33 2.2 (1.2,4.3) 2x/2x 35 403.7 (169.1, 963.6)  36 1722.2 (723.4, 4099.9) 35 4.2(2.0, 8.6) 4x/23vPS 30 488.9 (197.4, 1210.5) 30 831.7 (337.4, 2050.1) 301.7 (1.0, 2.8) 4x/4x 32 789.6 (392.0, 1590.7) 33 5496.3 (3414.4,8847.6)  32 6.9  (3.3, 14.4) 23vPS/7vPnC 62 88.5 (46.4, 168.9) 63 458.7(225.7, 931.9)  62 5.5  (2.9, 10.5) ^(a)Protocol specified timing forblood sample. ^(b)N = Number of subjects with assay results for thespecified serotype and the given visit. ^(c)Geometric Mean Titers (GMTs)were calculated using all subjects with available data for the specifiedblood draw. ^(d)Confidence limits (CI) are back transforms of aconfidence interval based on Student's t-distribution for the meanlogarithm of the concentrations or fold-rises. ^(e)Geometric Mean FoldRises (GMFRs) were calculated using all subjects with available datafrom both the pre-dose and post-dose blood draws. 2x = 9vPnCreconstituted with 7vPnC. 4x = 2 doses 9vPnC reconstituted with 2 doses7vPnC.

After an initial 7vPnC vaccine dose, geometric mean titers (GMTs) of sixout of seven serotypes assessed, were superior as compared to an initialdose of 23vPS vaccine. 7vPnC/23vPS recipients had higher GMTs (pointestimates) compared to 23vPS alone. 19F was the only serotype which didnot demonstrate superior activity response, however, the results did notdemonstrate activity of 19F serotype response to be inferior to othertreatment groups. See Table 11. In contrast, treatment group 23vPS/7vPnCrecipients, receiving a second dose of 7vPnC following an initial doseof 23vPS vaccine yielded lower GMTs as compared to 7vPnC alone for eachof the serotypes assessed. See Table 11.

The collection period for local and systemic reactogenicity after dose 1was 7 days. The protocol was amended to extend the collection period upto 14 days after dose 2. See Table 12 and Table 13 for a summary of theresults from dose 2. There were no serious adverse events that wereassessed to be vaccine related.

Pain at the injection site and redness were the most frequently observedlocal reactions after dose 1 and 2. Following dose 1, pain at theinjection site and redness were comparable between the 7vPnC and 23vPSgroups with a trend toward higher responses in the 7vPnC group.Following dose 2, more individuals reported pain and redness at theinjection site after polysaccharide following 7vPnC vaccine than afterpolysaccharide vaccine alone. See Table 12.

The incidences of any systemic reactions were comparable between 7vPnCvaccine and 23vPS vaccine after a single dose as shown in Example 1.Systemic reactions were higher (except fever) when 23vPS was given after7vPnC as compared to a single dose of either individual vaccine. Feverwas rare with a total of 9 cases after dose 1 and 9 cases after dose 2.After dose 1 all 9 cases were between 38° C. and 39° C. (1 after 7vPnC,2 after 2×PnC, 3 after 4×PnC, 3 after 23vPS). After dose 2, 8 cases werebetween 38° C. and 39° C. (3 after 7vPnC/23vPS, 2 after 2×/2×, 3 after23vPS/7vPnC), 1 case of fever>39° C. after 7vPnC/23vPS and none over 40°C. See Table 13.

TABLE 12 Percent of Subjects Reporting Injection Site Events Within 14Days Following Dose 2 Actual Treatment Group (Dose 1/Dose 2) 7vPnC/7vPnC/ 2x/ 4x/ 23vPS/ 23vPS 7vPnC 23vPS 2x/2x 23vPS 4x/4x 7vPnC p- N^(a)% N^(a) % N^(a) % N^(a) % N^(a) % N^(a) % N^(a) % value^(b) Redness Any37 45.9 37 24.3 34 52.9 35 25.7 33 39.4 39 64.1 72 30.6 0.001Significant^(c) 34 17.6 36 11.1 32 34.4 35 14.3 31 22.6 36 41.7 68 10.30.002 >7 cm^(d) 33 3.0 35 5.7 32 12.5 35 2.9 30 3.3 34 2.9 66 1.5 0.331Swelling Any 36 36.1 35 28.6 36 47.2 36 25.0 33 27.3 38 44.7 70 27.10.204 Significant^(e) 34 14.7 34 14.7 32 34.4 36 11.1 32 12.5 37 35.1 677.5 0.003 >7 cm^(d) 32 3.1 34 8.8 31 9.7 36 0.0 32 3.1 35 2.9 65 0.00.047 Pain at injection site Any 38 52.6 38 42.1 36 58.3 37 43.2 32 40.640 57.5 71 26.8 0.012 Significant^(c) 37 18.9 37 8.1 33 27.3 36 5.6 3215.6 37 13.5 70 0.0 <.001 Any Injection Site 37 67.6 36 52.8 36 77.8 3756.8 32 65.6 39 84.6 71 49.3 0.002 Reaction^(f) ^(a)N represents thenumber of subjects with known values. ^(b)Fisher Exact test, two-sided,for percent of subjects among all treatment groups. ^(c)Significant wasdefined as having a diameter for the involved area >8 caliper units or4.0 cm, including subjects with local reactions >7.0 cm. ^(d)Forcomparability with Dose 2, since subjects with local reactions >7.0 cmat Dose 1 were encouraged not to return for Dose 2. ^(e)Significant wasdefined as interfering with limb movement. ^(f)Any injection sitereaction includes any pain, any swelling, and any redness. Subjects wereincluded in this category if they experienced an event or had ‘no’ forall days for all events (i.e., any missing value excludes the subjectunless they experienced an event). 2x = 9vPnC reconstituted with 7vPnC.4x = 2 doses 9vPnC reconstituted with 2 doses 7vPnC.

TABLE 13 Percent of Subjects Reporting Systemic Reactions Within 14 DaysFollowing Dose 2 Actual Treatment Group (Dose 1/Dose 2) 7vPnC/ 7vPnC/2x/ 4x/ 23vPS/ Systemic 23vPS 7vPnC 23vPS 2x/2x 23vPS 4x/4x 7vPnC p-Reaction N^(a) % N^(a) % N^(a) % N^(a) % N^(a) % N^(a) % N^(a) %value^(b) Fever ≧38° C. 36 8.3 38 0.0 34 0.0 35 5.7 34 0.0 38 0.0 68 5.90.112 Fever >39° C. 36 2.8 38 0.0 34 0.0 35 0.0 34 0.0 38 0.0 68 0.00.493 Fever >40° C. 36 0.0 38 0.0 34 0.0 35 0.0 34 0.0 38 0.0 680.0 >.999 Fatigue 38 42.1 40 25.0 35 37.1 36 27.8 34 5.9 39 17.9 71 28.20.009 Headache 38 23.7 40 12.5 36 19.4 36 16.7 34 2.9 39 7.7 72 16.70.147 Chills 37 18.9 39 10.3 34 5.9 36 5.6 34 5.9 40 7.5 71 7.0 0.492Rash 36 2.8 39 0.0 34 2.9 36 2.8 33 3.0 38 2.6 70 1.4 0.911 Vomiting 360.0 38 0.0 35 5.7 36 2.8 34 2.9 39 0.0 70 1.4 0.348 Decreased appetite36 8.3 38 7.9 34 8.8 36 8.3 34 8.8 39 12.8 70 11.4 >.999 Joint pain 3716.2 38 26.3 35 20.0 36 13.9 33 27.3 40 20.0 69 15.9 0.689 Aggravatedjoint 28 10.7 31 19.4 31 16.1 26 7.7 27 18.5 35 14.3 57 14.0 0.893 painNew joint pain 28 7.1 30 6.7 31 16.1 27 3.7 28 10.7 36 16.7 59 3.4 0.211Muscle pain 38 34.2 38 21.1 36 38.9 37 18.9 31 25.8 40 25.0 73 20.50.338 Aggravated muscle 31 12.9 30 6.7 33 12.1 26 3.8 26 7.7 32 6.3 557.3 0.885 pain New muscle pain 31 22.6 32 15.6 33 33.3 30 16.7 29 34.536 22.2 61 13.1 0.165 Medication to treat 33 3.0 37 0.0 31 0.0 33 6.1 280.0 33 3.0 61 1.6 0.568 fever Any new 33 12.1 35 2.9 30 6.7 32 6.3 267.7 32 12.5 52 5.8 0.738 medications Any systemic 37 67.6 36 55.6 3661.1 33 45.5 29 69.0 35 42.9 63 55.6 0.221 reaction^(c) ^(a)N representsthe number of subjects with known values. ^(b)Fisher Exact test,two-sided, for percent of subjects among all treatment groups. ^(c)Anysystemic reaction excludes any new medications and any medications totreat a fever. Subjects were included in this category if theyexperienced an event or had ‘no’ for all days for all events (i.e., anymissing value excludes the subject unless they experienced an event). 2x= 9vPnC reconstituted with 7vPnC. 4x = 2 doses 9vPnC reconstituted with2 doses 7vPnC.

Summary of Study Results

This study demonstrated that conjugate pneumococcal vaccine induced avigorous immune response for all vaccine serotypes after dose 1.Antibody levels were superior for all conjugate serotypes (except 19F,which was non-inferior) relative to unconjugated polysaccharide vaccine.Functional antibody activity in subjects receiving conjugate vaccine wasalso superior to that induced by polysaccharide vaccine after dose 1.Safety and tolerability were comparable between conjugate andpolysaccharide.

To assess whether an earlier dose of conjugate vaccine could complementa subsequent dose of polysaccharide (7vPnC/23vPS), antibody responsesafter a second polysaccharide vaccine dose were compared to antibodylevels after a single dose of polysaccharide vaccine (standard of care).For all common serotypes (except serotype 4), antibody levels trendedhigher than after polysaccharide alone. For all common serotypes,functional antibody activity resulted higher levels than afterunconjugated polysaccharide vaccine alone. This suggests that conjugatevaccine improved the response to a subsequent dose of polysaccharidevaccine.

By contrast, individuals who were vaccinated with unconjugatedpolysaccharide first, before receiving conjugate (23vPS/7vPnC), hadlower antibody levels after the subsequent conjugate compared to asingle dose of conjugate. These observations support previous dataindicating that the 23vPS vaccine may induce hyporesponsiveness.

The antibody response to a second dose of conjugate (7vPnC/7vPnC)trended higher than after a single dose of unconjugated polysaccharidefor the majority of serotypes. Compared to the initial dose ofconjugate, the immune response after the subsequent dose of conjugatevaccine was similar.

Local and systemic reactogenicity was comparable between conjugate andunconjugated polysaccharide after dose 1, with a trend toward higherresponses in the 7vPnC group. A subsequent dose of polysaccharideincreased local and systemic reactogenicity (except fever) relative to asingle dose of conjugate or unconjugated polysaccharide. However, themajority of adverse events were non serious and were of short duration.There were no vaccine-related serious adverse events. The overall safetyprofile was acceptable.

Example 3 Comparison of Activity Levels of Pneumococcal Antibodies inOlder Adult and Infant Populations

Conjugate vaccine has been shown to significantly decrease pneumococcalpneumonia in three separate studies in infants. (S. Black, et al, Eur JPediatr. 161 Suppl 2:S127-31. 2002; K. Klugman, et al, N Engl J Med.349:1341-8. 2003; and F. Cutts, et al, Lancet. 365:1139-46, 2005.) Thecurrent 23vPs vaccine does not protect against community acquiredpneumococcal pneumonia (CAP) in elderly adults. Antibody responsesobtained in older adult populations described in Examples 1 and 2 abovewere compared to responses in infants after three doses of 7vPnC at two,four, and six months. The results are shown in Table 14.

The antibody response to a single dose of conjugate (7vPnC) in olderadults induced OPA levels similar to those of infants after three dosesof 7vPnC. See Table 14. In contrast, immunization with unconjugatedpolysaccharide induced increased levels of IgG antibodies, but themagnitude of the OPA titer is significantly lower than those achievedafter immunization with 7vPnC. See Table 14. These results supportconjugate vaccine achieved levels which may be able to induce protectionin the elderly against pneumococcal pneumonia.

TABLE 14 Pneumococcal Antibody Levels in Elderly Subjects immunized withone dose of 7 Valent PnC or 23valent Ps vs. Infants Immunized with 3doses of 7 valent PnC Infants Elderly Elderly (post 3 (post 1 (post 1doses PnC) dose PnC) dose 23vPs) Type ELISA OPA ELISA OPA ELISA OPA  43.29 1571 3.27 1504 1.4 669.9  6B 5.18 1888 8.02 1351.2 4.56 809.2  9V1.88 3551 9.82 2914.6 3.63 984.6 14 7.13 3017 17.5 2165 8.5 974.5 18C2.88 1559 12.97 1317.5 6.82 464.6 19F 4.17 203 5.5 182.2 4.43 202.7 23F2.16 4845 12.4 1309.3 3.97 302.4

One skilled in the art will readily ascertain the essentialcharacteristics of the invention, and understand that the foregoingdescription and examples are illustrative of practicing the providedinvention. Those skilled in the art will be able to ascertain using nomore than routine experimentation, many variations of the detailpresented herein may be made to the specific embodiments of theinvention described herein without departing from the spirit and scopeof the present invention.

Patents, patent applications, publications, and the like are citedthroughout the application. The disclosures of each of these documentsare incorporated herein by reference in their entirety.

1-26. (canceled)
 27. A method of eliciting an immunoprotective antibodyresponse to a capsular polysaccharide of Streptococcus pneumoniae in anolder subject, comprising administering to a older subject animmunogenic amount of an initial pneumococcal polysaccharide conjugatevaccine, wherein the subject has previously received a pneumococcalunconjugated polysaccharide vaccine, and wherein the prioradministration of pneumococcal vaccine was administered at an intervalselected from the group consisting of at least one year, at least twoyears and at least five years prior to administration of the initialpneumococcal polysaccharide conjugate vaccine administration.
 28. Themethod of claim 27, further comprising administering at least oneadditional vaccine dose.
 29. The method of claim 28, wherein theadditional dose of vaccine is selected from the group consisting of apneumococcal polysaccharide conjugate vaccine composition, anunconjugated pneumococcal polysaccharide vaccine composition, andcombinations thereof.
 30. The method of claim 28, wherein a second doseof vaccine is administered at a time selected from the group consistingof about two weeks, about one month, about six weeks, about two months,about three months, about six months, about nine months, about twelvemonths, about fifteen months, about eighteen months, about twenty-onemonths and about twenty four months following the initial conjugatevaccine administration.
 31. The method of claim 28, wherein at least twoadditional vaccine doses are administered following the initialconjugate dose and wherein the third or later additional doses areadministered at intervals comprising at least about one year after theprior dose.
 32. The method of claim 31, wherein additional vaccineadministration dosed in an administration interval selected from oneyear, five years, and ten years from a prior dose.
 33. The method ofclaim 32, wherein the third or later dose of vaccine comprises apneumococcal polysaccharide conjugate vaccine.
 34. The method of claim32, wherein the third or later dose of vaccine comprises an unconjugatedpneumococcal polysaccharide vaccine composition.
 35. The method of claim27, wherein the conjugated vaccine is selected from the group consistingof a 7-valent, 9-valent, 10-valent, 11-valent and 13-valent vaccineconjugate vaccine.
 36. The method of claim 27, wherein the unconjugatedpolysaccharide vaccine is a 23-valent pneumococcal polysaccharidevaccine.
 37. The method of claim 27, wherein the conjugate vaccinecomprises polysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.38. The method of claim 27 wherein the conjugated vaccine comprisespolysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F.39. The method of claim 27 wherein the conjugated vaccine comprisespolysaccharides of serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and23F.
 40. The method of claim 27 wherein the conjugated vaccine comprisespolysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9A, 9V, 14, 18C,19F, and 23F.
 41. The method of claim 27, wherein the polysaccharidesare conjugated to a polypeptide carrier.
 42. The method of claim 41,wherein the polypeptide carrier is selected from the group consisting ofthe meningococcal outer membrane protein complex, the CRM₁₉₇ variant ofdiphtheria toxin, diphtheria toxoid, tetanus toxoid, cholera toxoid,pertussis toxoid, inactivated or mutant pneumococcal pneumolysin,pneumococcal surface protein A or a derivative thereof, pneumococcaladhesion protein A or a derivative thereof, C5a peptidase group a orgroup b streptococcus or a derivative thereof, non-typable H. influenzaeP4 protein or a derivative thereof, non-typable H. influenzae P6 proteinor a derivative thereof, M. catarrhalis uspA or a derivative thereof,Keyhole Limpet Haemocyanin (KLH), protein derivative of Tuberculin(PPD), protein D from H. influenzae, OMPC of N. meningitidis.
 43. Themethod of claim 35 wherein the conjugated vaccine comprisespolysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F conjugatedto the non-toxic CRM₁₉₇ variant of diphtheria toxin.
 44. The method ofclaim 36 wherein the unconjugated vaccine is PNEUMOVAX®23 pneumococcalpolysaccharide vaccine.
 45. The method of claim 36 wherein theunconjugated vaccine comprises polysaccharides of serotypes 1, 2, 3, 4,5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F, and 33F.
 46. The method of claim 33, wherein the conjugatevaccine comprises polysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F,and 23F.
 47. The method of claim 33 wherein the conjugated vaccinecomprises polysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C,19F, and 23F.
 48. The method of claim 33 wherein the conjugated vaccinecomprises polysaccharides of serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C,19F, and 23F.
 49. The method of claim 33 wherein the conjugated vaccinecomprises polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9A, 9V,14, 18C, 19F, and 23F.
 50. The method of claim 33 wherein thepolysaccharides are conjugated to a polypeptide carrier.
 51. The methodof claim 50, wherein the polypeptide carrier is selected from the groupconsisting of the meningococcal outer membrane protein complex, theCRM₁₉₇ variant of diphtheria toxin, diphtheria toxoid, tetanus toxoid,cholera toxoid, pertussis toxoid, inactivated or mutant pneumococcalpneumolysin, pneumococcal surface protein A or a derivative thereof,pneumococcal adhesion protein A or a derivative thereof, C5a peptidasegroup a or group b streptococcus or a derivative thereof, non-typable H.influenzae P4 protein or a derivative thereof, non-typable H. influenzaeP6 protein or a derivative thereof, M. catarrhalis uspA or a derivativethereof, Keyhole Limpet Haemocyanin (KLH), protein derivative ofTuberculin (PPD), protein D from H. influenzae, OMPC of N. meningitidis.52. The method of claim 46 wherein the conjugated vaccine comprisespolysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F conjugatedto the non-toxic CRM₁₉₇ variant of diphtheria toxin. 53-105. (canceled)